Compositions and methods for making selenocysteine containing polypeptides

ABSTRACT

Non-naturally occurring tRNA Sec  and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA Sec  can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA Sec  a recombinant expression system, such as  E. coli , with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA Sec .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/131,382, which isa 371 application of the International Application No. PCT/US2012/046252entitled “Compositions and Methods for Making Selenocysteine ContainingPolypeptides”, filed in the United States Receiving Office for the PCTon Jul. 11, 2012, which claims the benefit of and priority to U.S.Provisional Application No. 61/506,338, entitled “System forco-translational selenocysteine insertion at any position of a protein”filed Jul. 11, 2011 which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Agreement Nos.GM022854 awarded by National Institute of Health, DE-FG02-98ER20311awarded by the Department of Energy and 0950474 awarded by the NationalScience Foundation. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted as a text file named “YU_5714_ST25.txt,”created on Jan. 7, 2014, and having a size of 15,578 bytes is herebyincorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The field of the invention generally relates to compositions includingrecombinant tRNAs and methods of using them to manufacture recombinantselenocysteine containing polypeptides.

BACKGROUND OF THE INVENTION

Selenocysteine, commonly referred to as the twenty-first amino acid, isincorporated into at least 25 human proteins. Natural co-translationalincorporation of selenocysteine (Sec) into proteins proceeds by arecoding process so that upon encountering the UGA codon in themessenger RNA the ribosome knows to recognize it as Sec instead of Stop.This process requires three components: (i) the aminoacyl-tRNA carryingselenocysteine, Sec-tRNA^(Sec); (ii) the specialized elongation factor,SelB, carrying Sec-tRNA^(Sec) to the ribosome, and (iii) the SECISelement, an RNA secondary structure of the mRNA just downstream of theUGA codon, that interacts with the SelB•Ser-tRNA^(Sec) complex (Bock, A,Thanbichler, M, Rother, M & Resch, A (2005), eds Ibba M, Francklyn C S,& Cusack S (Landes Bioscience, Georgetown, Tex.), pp 320-327; Yoshizawa,S & Bock, A (2009) Biochim Biophys Acta 1790:1404-1414). Additionally,in order to protect the integrity of this recoding process,Sec-tRNA^(Sec) is not recognized by the general elongation factor EF-Tubecause of the presence of three base pairs that act as antideterminants(Rudinger, J, Hillenbrandt, R, Sprinzl, M & Giegé, R (1996) EMBO J15:650-657. Sec-tRNA^(Sec) cannot be accommodated during normaltranslation because it is not an acceptable substrate for EF-Tu, and theSelB•Sec-tRNA^(Sec) complex will not decode in-frame UGA codons inabsence of the SECIS.

Insertion of selenocysteine into a recombinant protein, for example,substitution of a naturally occurring cysteine residue forselenocysteine, can alter the function of the protein. Substituting oneor more naturally occurring Cys residues in the active site of an enzymewith a Sec can increase the activity of this enzyme. Diselenide bondshave very low redox potential. Therefore, replacing disulfide bonds withdiselenide or selenocysteine-cysteine bonds can lower dosage, increasehalf-life, increase stability, reduce toxicity, alter pharmacokinetics,change folding properties, or combinations thereof of the recombinantselenocysteine containing protein relative to a reference proteinwithout selenocysteines, such as a naturally occurring counterpart.

However, due the presence the SECIS element as an integral part of theopen reading frame (within the mRNA) encoding the protein that harborsSec in its sequence, it is not possible to insert Sec into proteins by astandard mutational scheme or in the construction of random mutageniclibraries, and production of Sec proteins is limited to costly andinefficient methods of protein synthesis. Accordingly, there is a needfor alternative methods of manufacturing selenocysteine containingpolypeptides.

It is an object of the invention to provide compositions and methods forrecombinant expression of proteins engineered to include one or moreselenocysteine residues without the requirement of a SECIS in the mRNAencoding the protein.

It is a further object of the invention to provide non-naturallyoccurring proteins including one or more selenocysteine residues.

SUMMARY OF THE INVENTION

Non-naturally occurring tRNA^(Sec) and methods of using them forrecombinant expression of proteins engineered to include one or moreselenocysteine residues are disclosed. Typically, the non-naturallyoccurring tRNA^(Sec) (1) can be recognized by SerRS and by EF-Tu, orvariants thereof; and is characterized by one or more of the followingelements: (2) when aminoacylated with serine, the non-naturallyoccurring Ser-tRNA^(Sec) can be converted to non-naturally occurringSec-tRNA^(Sec) by SelA, or variant thereof; (3) when aminoacylated withserine, the non-naturally occurring Ser-tRNA^(Sec) can be phosphorylatedby PSTK or variant thereof; (4) when aminoacylated with phosphorylatedserine, the non-naturally occurring Sep-tRNA^(Sec) can serve as asubstrate for SepSecS or variant thereof; and combinations thereof. Insome embodiments, the non-naturally occurring Ser-tRNA^(Sec) ischaracterized by elements (1) and (2). In some embodiments, thenon-naturally occurring Ser-tRNA^(Sec) is characterized by elements (1),(3), and (4). In some embodiments, the non-naturally occurringSer-tRNA^(Sec) is characterized by elements (1), (2), (3), and (4). Insome embodiments, the non-naturally occurring Ser-tRNA^(Sec) ischaracterized by elements (1), (2), and (3).

The non-naturally occurring tRNA^(Sec) do not require a SECIS element inan mRNA to be incorporated into a growing polypeptide chain duringtranslation. Typically the anticodon of the non-naturally occurringtRNA^(Sec) is recognized or hybridizes to a stop codon.

Methods of manufacturing selenocysteine containing polypeptides are alsodisclosed. The non-naturally occurring tRNA^(Sec) can be used forrecombinant manufacture of selenocysteine containing polypeptidesencoded by mRNA without the requirement of an SECIS element. In someembodiments, the non-naturally occurring tRNA^(Sec) is co-expressed in arecombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA,or PSTK and SepSecS, or a combination of SelA, PSTK and SepSecS, and anmRNA with at least one codon that recognizes the anticodon of thenon-naturally occurring tRNA^(Sec) to manufacture a selenocyteinecontaining polypeptide encoded by the mRNA.

Nucleic acids encoding selenocysteine containing polypeptides are alsodisclosed. The nucleic acids encode a polypeptide of interest andinclude a non-natural tRNA^(Sec) recognition codon, for example a “stop”codon, that hybridizes with the anticodon of the non-naturally occurringtRNA^(Sec), such that a selenocyteine is transferred onto the growingpolypeptide chain during translation. The selenocysteine containingpolypeptides can be polypeptides that contain selenocysteine in nature,or polypeptides that do not contain selenocysteine in nature. Forexample, a non-naturally occurring tRNA recognition codon can besubstituted for a cysteine codon in the naturally occurring mRNA, whichchanges the cysteine to a selenocysteine when the nucleic acid encodingthe polypeptide is expressed recombinantly with the non-naturallyoccurring tRNA^(Sec) Substituting one or more naturally occurring Cysresidues with a Sec can increase activity, lower dosage, reducetoxicity, improve stability, increase efficacy, increase half-life orcombinations thereof of a selenocysteine containing protein relative toits cysteine containing counterpart.

Methods of treating subjects in need thereof with recombinantselenocysteine containing polypeptides prepared using the disclosedcompositions and methods are also disclosed. Particularly preferredproteins containing selenocystein include antibodies and enzymes havingaltered binding affinity and/or pharmacokinetics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are illustrations showing the canonical and Sec translationapparatuses respectively. The canonical amino acids are charged ontotheir respective tRNA by their cognate aminoacyl-tRNA synthetase. Theaminoacyl-tRNA is then delivered by EF-Tu to the ribosome (FIG. 1A). Incontrast, the Sec pathway requires several biosynthetic steps. First,tRNA^(Sec) is misacylated to Ser-tRNA^(Sec) by SerRS. While in bacteriaSer-tRNA^(Sec) is directly converted by SelA to Sec-tRNA^(Sec), archaeaand eukaryotes employ an additional phosphorylation step by PSTK to formSep-tRNA^(Sec), which is then converted by SepSecS to the final productSec-tRNA^(Sec) (FIG. 1B). Sec-tRNA^(Sec) is bound by elongation factorSelB and delivered to the ribosome. However, reassignment of the opalcodon UGA to a Sec codon is only achieved if SelB also binds to the mRNASECIS hairpin structure.

FIG. 2 is a depiction of the primary and secondary structures of humantRNA^(Sec) (SEQ ID NO:3) adapted from Yuan, et al., FEBS Lett.,584(2):342-349 (2010).

FIG. 3 is a depiction of the primary and secondary structures of E. colitRNA^(Sec) (left, SEQ ID NO: 1), a non-naturally occurring tRNA^(UTu)with an E. coli body (center, (tRNA^(UTu) _(op), SEQ ID NO:6; tRNA^(Utu)_(am), SEQ ID NO:7), and E. coli tRNA^(Ser) (right. SEQ ID NO:4). E.coli tRNA^(Ser) (right) serves as a major scaffold for tRNA^(Utu)(center) with the exception of the acceptor stem that originates from E.coli tRNA^(Sec) (boxed sequence elements). Major EF-Tu recognitionelements were retained from tRNA^(Ser) as well (circled sequenceelements). Substitution of the amber anti-codon CUA (tRNA^(UTu) _(am))for the opal anti-codon UCA (tRNA^(UT) _(op)) are depicted with arrowsand labeling.

FIGS. 4A and 4B are depictions of the primary and secondary structuresof a non-naturally occurring tRNA^(UTu) with a body derived from M.maripaludis (FIG. 4A, tRNA^(Utu) _(UCA), SEQ ID NO:57; tRNA^(UTu) _(op),SEQ ID NO:33; tRNA^(Uta) _(am), SEQ ID NO:34) and a non-naturallyoccurring tRNA^(UTu) with a body derived from E. coli (FIG. 4B,tRNA^(Utu) _(UCA), SEQ ID NO:58; tRNA^(UTu) _(op), SEQ ID NO:9;tRNA^(Uta) _(am), SEQ ID NO:10). Transplanted PSTK identity elements areboxed. “<” identifies potential locations of additional base pairs inthe acceptor stem. “Arrow” identifies the location of other possiblemutations. Specifically, the <depict one possible insertion of a G-Cbase pair between the 1^(st) and 2^(nd) base pair and a second possibleinsertion of a G-C pair insertion between the 6^(th) and 7^(th) basepair of the acceptor stem. The arrows depict a possible change in the50:64 base pair (A-U) to a U-A pair, and substitution of the serineanticodon (UGA) with opal (UCA) or amber (CUA) anticodon.

FIG. 5 is a photograph showing FDH_(H) activity in E. coli MH5 (selA,selB, fdhF mutant) strain transformed with (clockwise from top) (1)SelA+PSTK+SelB+FDH_(H)(UGA); (2) SelA+PSTK+FDH_(H)(UAG); (3)SelA+PSTK+tRNA^(UTu)(amber)+FDH_(H)(UAG); (4) empty plasmid.

FIG. 6 is a photograph showing thymidylate synthase activity in E. coliMH6 (selA, selB, thyA mutant) strain transformed with (clockwise fromtop) (1) tRNA^(UTu)(amber)+SelA+PSTK+thyA (wildtype); (2)tRNA^(UTu)(amber)+SelA+PSTK+thyA (Cys146Ser); (3)tRNA^(UTu)(amber)+SelA+PSTK+thyA (Cys146amber); (4) empty plasmid; grownin M9+1 μM Na₂SeO₃+5 μM IPTG+thymine (20 μg/ml) (left panel) or M9+1 μMNa₂SeO₃+5 μM IPTG+w/o thymine (right panel).

FIG. 7A is a line graph showing the disulfide oxidoreductase activity(V₀(mMs⁻¹)) of Grx1_(C11am/C14S)Sec, Grx1_(C11S/C14S) and Grx1_(C14S) atincreasing concentrations (nM). FIG. 7B is a line graph showing theperoxidase activity (V₀[cat]⁻¹(s⁻¹)) of Grx1_(C11am/C14S)Sec,Grx1_(C11/C14S) and Grx1_(C14S) as a function of reduced glutathioneconcentration at 25° C. (mM). FIG. 7C is a line graph showing peroxidaseactivity (units (mL⁻¹)) of Sec containing GPx1_(am) and Cys containingGPx1_(Cys) that were overexpressed in E. coli and compared tocommercially available GPx_(hum) from human erythrocytes as a functionof GPx1 (10⁻³ mg mL⁻¹).

FIG. 8 is a spectrogram showing the presence of selenocysteine at aminoacid position 11 in Grx1_(C11am/C14S)Sec by mass spectroscopy. Shown isthe MS/MS spectrum of the trypsin-digested Sec-containing fragmentS₉G₁₀U₁₁P₁₂Y₁₃S₁₄V₁₅R₁₆. Fragments observed in the second massspectrometric analysis of this peptide are labeled with b3, y2, y3, y4and y5. The Sec residue of the peptide in the MS/MS experiment was firsttreated with DTT, and then alkylated with iodoacetamide for oxidativeprotection of the selenol. The unit m/z describes the mass-to-chargeratio.

FIGS. 9A and 9B are spectrograms showing the results of Fouriertransform ion cyclotron resonance (FT-ICR) mass spectrometry ofGrx1_(C11S/C14S) (calculated: 10,650 Da. found 10,651 Da) andGrx1_(C11am/C14S) (calculated: 11,019 Da. found 11,019 Da).

FIG. 10 is a spectrogram showing the results of Fourier transform ioncyclotron resonance (FT-ICR) mass spectrometry of GPx1am.

FIG. 11A is a line graph showing varying concentrations (1-30 μM) oftRNA^(Ser) (--), tRNA^(Sec) (-▪-), and tRNA^(Utu) (-♦-) in an assay formeasuring kinetic parameter of each tRNA as a substrate for SerRS (v[μMaa-tRNAmin⁻¹]). Kinetic parameters were determined by Michaelis & Mentenplots of the initial aminoacylation velocity versus substrateconcentration. FIG. 11B is a line graph showing in vitro conversion oftRNA^(Sec) (-♦-) and tRNA^(UTu) (-▪-) (Ser-tRNA to Sec-tRNA %) by SelAas a function of time (min).

FIG. 12 is a line graph showing in vitro conversion of tRNA^(Sec) (-♦-)and tRNA^(UTu) (-▪-) (Ser-tRNA to Sep-tRNA %) by PSTK as a function oftime (min).

FIG. 13A is a structure diagram showing the activity of variousO-6-methylguanine-DNA methyltransferase (MGMT) constructs onO6-methylguanine. FIG. 13B is an image showing the activity (in numberof living colonies) of E. coli Δada Δotg-1 cells expressing either MGMTC145, amber145 (Sec/Ser) or S145 mutant proteins, and tRNA^(UTu)amber,pulsed 3× with N-Methyl-N-nitroso-N′-nitroguanidine.

FIG. 14 is a diagram showing genomic factors in E. coli that increaseSec incorporation into the recombinantly expressed selenocysteinecontaining protein Grx (%).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Transfer RNA or tRNA refers to a set of genetically encoded RNAs thatact during protein synthesis as adaptor molecules, matching individualamino acids to their corresponding codon on a messenger RNA (mRNA). Inhigher eukaryotes such as mammals, there is at least one tRNA for eachof the 20 naturally occurring amino acids. In eukaryotes, includingmammals, tRNAs are encoded by families of genes that are 73 to 150 basepairs long. tRNAs assume a secondary structure with four base pairedstems known as the cloverleaf structure. The tRNA contains a stem and ananticodon. The anticodon is complementary to the codon specifying thetRNA's corresponding amino acid. The anticodon is in the loop that isopposite of the stem containing the terminal nucleotides. The 3′ end ofa tRNA is aminoacylated by a tRNA synthetase so that an amino acid isattached to the 3′end of the tRNA. This amino acid is delivered to agrowing polypeptide chain as the anticodon sequence of the tRNA reads acodon triplet in an mRNA.

As used herein “suppressor tRNA” refers to a tRNA that alters thereading of a messenger RNA (mRNA) in a given translation system. Forexample, a suppressor tRNA can read through a stop codon.

As used herein, an “anticodon” is any combination of 2, 3, 4, and 5bases (G or A or U or C) that are complementary to a “stop codon” ofequivalent and complementary base composition. Known “stop codons”include, but are not limited to, the three codon bases, UAA known asochre, UAG known as amber and UGA known as opal that do not code for anamino acid but act as signals for the termination of protein synthesis.Generally the anticodon loop consists of seven nucleotides. In the 5′ to3′ direction the first two positions 32 and 33 precede the anticodonpositions 34 to 36 followed by two nucleotides in positions 37 and 38(Alberts, B., et al. in The Molecular Biology of the Cell, 4^(th) ed,Garland Science, New York, N.Y. (2002)). The size and nucleotidecomposition of the anticodon is generally the same as the size of thecodon with complementary nucleotide composition. A four base pair codonconsists of four bases such as 5′-AUGC-3′ and an anticodon for such acodon would complement the codon such that the tRNA contained 5′-GCAU-3′with the anticodon starting at position 34 of the tRNA. A 5 base codon5′-CGGUA-3′ codon is recognized by the 5′-UACCG-3′ anticodon (HohsakaT., et al. Nucleic Acids Res. 29:3646-3651 (2001)). The composition ofany such anticodon for 2 (16=any possible combination of 4 nucleotides),3 (64), 4 (256), and 5 (1024) base codons would follow the same logicalcomposition. The “anticodon” typically starts at position 34 of acanonical tRNA, but may also reside in any position of the “anti-codonstem-loop” such that the resulting tRNA is complementary to the “stopcodon” of equivalent and complementary base composition.

As used herein, “tRNA^(Sec)” refers to an unaminoacylated tRNA suitablefor carrying selenocysteine. Typically the anticodon sequence of thetRNA^(Sec) can recognize or hybridize with an mRNA codon specific for,or designed to encode, a selenocysteine amino acid, for example UGA. InE. coli, the endogenous tRNA^(Sec) is encoded by the selC gene.

As used herein, “tRNA^(Ser)” refers to an unaminoacylated tRNA suitablefor carrying serine. Typically the anticodon sequence of the tRNA^(Ser)can recognize or hybridize with an mRNA codon specific for, or designedto encode, a serine amino acid, for example UCU, UCC, UCA, UCG, AGU, orAGC.

As used herein, “tRNA^(UTu)” refers to a non-naturally occurring,unaminoacylated tRNA^(Sec) suitable for carrying selenocysteine.Typically the anticodon sequence of the tRNA^(UTu) can recognize orhybridize with an mRNA codon specific for, or designed to encode, aselenocysteine amino acid.

As used herein, “Sec-tRNA^(Sec)” refers to aminoacylated tRNA^(Sec)carrying a selenocysteine amino acid.

As used herein, “Ser-tRNA^(Sec)” refers to aminoacylated tRNA^(Sec)carrying a serine amino acid.

As used herein, “Ser-tRNA^(Ser)” refers to aminoacylated tRNA^(Ser)carrying a serine amino acid.

As used herein, “Sep-tRNA^(Ser)” refers to a phosphorylatedSer-tRNA^(Sec).

As used herein, “EF-Tu” refers to Elongation Factor Thermo Unstable, aprokaryotic elongation factor mediates the entry of the aminoacyl-tRNAinto a free site of the ribosome.

As used herein, “SerRS” refers to Seryl-tRNA synthetase (also known asSerine—tRNA ligase) which is a prokaryotic factor that catalyzes theattachment of serine to tRNA^(Ser).

As used herein “SECIS” refers to a SElenoCysteine Insertion Sequence, isan RNA element around 60 nucleotides in length that adopts a stem-loopstructure which directs the cell to translate UGA codons asselenocysteines. In bacteria the SECIS can be soon after the UGA codonit affects, while in archaea and eukaryotes, it can be in the 3′ or 5′UTR of an mRNA, and can cause multiple UGA codons within the mRNA tocode for selenocysteine.

As used herein “SelA” refers to selenocysteine synthase, a prokaryoticpyridoxal 5-phosphate-containing enzyme which catalyzes the conversionof Ser-tRNA^(Sec) into a Sec-tRNA^(Sec).

As used herein “SelB” refers to selenocysteine-specific elongationfactor, a prokaryotic elongation factor for delivery of Sec-tRNA^(Sec)to the ribosome.

As used herein “PSTK” refers to phosphoseryl-tRNA kinase (also known asO-phosphoseryl-tRNA^(Sec) kinase and L-seryl-tRNA^(Sec) kinase), akinase that phosphorylates Ser-tRNA^(Sec) to O-phosphoseryl-tRNA^(Sec),an activated intermediate for selenocysteine biosynthesis.

As used herein “SepSecS” refers to Sep (O-phosphoserine) tRNA: Sec(selenocysteine) tRNA synthase (also known as O-phosphoseryl-tRNA(Sec)selenium transferase and Sep-tRNA:Sec-tRNA synthase), an eukaryotic andarchaeal enzyme that converts O-phosphoseryl-tRNA^(Sec) toselenocysteinyl-tRNA^(Sec) in the presence of a selenium donor.

As used herein SepCysS refers to Sep-tRNA:Cys-tRNA synthase, anarchaeal/eukaryotic enzyme that converts O-phosphoseryl-tRNA^(Cys)(Sep-tRNA^(Cys)) into Cys-tRNA^(Cys) in the presence of a sulfur donor.

As used herein “G-C content” (or guanine-cytosine content) refers to thepercentage of nitrogenous bases on a nucleic acid molecule, or fragment,section, or region thereof, that are either guanine or cytosine.

Aminoacyl-tRNA Synthetases (“AARS”) are enzymes that charge (acylate)tRNAs with amino acids. These charged aminoacyl-tRNAs then participatein mRNA translation and protein synthesis. The AARS show highspecificity for charging a specific tRNA with the appropriate aminoacid, for example, tRNA^(Val) with valine by valyl-tRNA synthetase ortRNA^(Trp) with tryptophan by tryptophanyl-tRNA synthetase. In general,there is at least one AARS for each of the twenty amino acids.

As used herein “translation system” refers to the components necessaryto incorporate a naturally occurring amino acid into a growingpolypeptide chain (protein). Components of a translation system caninclude, e.g., ribosomes, tRNAs, synthetases, mRNA and the like. Thecomponents described herein can be added to a translation system, invivo or in vitro. A translation system can be either prokaryotic, e.g.,an E. coli cell, or eukaryotic, e.g., a yeast, mammal, plant, or insector cells thereof.

A “transgenic organism” as used herein, is any organism, in which one ormore of the cells of the organism contains heterologous nucleic acidintroduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. Suitabletransgenic organisms include, but are not limited to, bacteria,cyanobacteria, fungi, plants and animals. The nucleic acids describedherein can be introduced into the host by methods known in the art, forexample infection, transfection, transformation or transconjugation.Techniques for transferring DNA into such organisms are widely known andprovided in references such as Sambrook, et al. (2000) MolecularCloning: A Laboratory Manual, 3^(rd) ed., vol. 1-3, Cold Spring HarborPress, Plainview N.Y.

As used herein, the term “eukaryote” or “eukaryotic” refers to organismsor cells or tissues derived therefrom belonging to the phylogeneticdomain Eukarya such as animals (e.g., mammals, insects, reptiles, andbirds), ciliates, plants (e.g., monocots, dicots, and algae), fungi,yeasts, flagellates, microsporidia, and protists.

As used herein, the term “non-eukaryotic organism” refers to organismsincluding, but not limited to, organisms of the Eubacteria phylogeneticdomain, such as Escherichia coli, Thermus thermophilus, and Bacillusstearothermophilus, or organisms of the Archaea phylogenetic domain suchas, Methanocaldococcus jannaschii, Methanothermobacterthermautotrophicus, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, and Aeuropyrum pernix.

The term “construct” refers to a recombinant genetic molecule having oneor more isolated polynucleotide sequences. Genetic constructs used fortransgene expression in a host organism include in the 5′-3′ direction,a promoter sequence; a sequence encoding a gene of interest; and atermination sequence. The construct may also include selectable markergene(s) and other regulatory elements for expression.

The term “gene” refers to a DNA sequence that encodes through itstemplate or messenger RNA a sequence of amino acids characteristic of aspecific peptide, polypeptide, or protein. The term “gene” also refersto a DNA sequence that encodes an RNA product. The term gene as usedherein with reference to genomic DNA includes intervening, non-codingregions as well as regulatory regions and can include 5′ and 3′ ends.

The term “orthologous genes” or “orthologs” refer to genes that have asimilar nucleic acid sequence because they were separated by aspeciation event.

The term polypeptide includes proteins and fragments thereof. Thepolypeptides can be “exogenous,” meaning that they are “heterologous,”i.e., foreign to the host cell being utilized, such as human polypeptideproduced by a bacterial cell. Polypeptides are disclosed herein as aminoacid residue sequences. Those sequences are written left to right in thedirection from the amino to the carboxy terminus. In accordance withstandard nomenclature, amino acid residue sequences are denominated byeither a three letter or a single letter code as indicated as follows:Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid(Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V).

“Cofactor”, as used herein, refers to a substance, such as a metallicion or a coenzyme that must be associated with an enzyme for the enzymeto function. Cofactors work by changing the shape of an enzyme or byactually participating in the enzymatic reaction.

“Variant” refers to a polypeptide or polynucleotide that differs from areference polypeptide or polynucleotide, but retains essentialproperties. A typical variant of a polypeptide differs in amino acidsequence from another, reference polypeptide. Generally, differences arelimited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more modifications (e.g., substitutions, additions, and/ordeletions). A substituted or inserted amino acid residue may or may notbe one encoded by the genetic code. A variant of a polypeptide may benaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally.

Modifications and changes can be made in the structure of thepolypeptides of in disclosure and still obtain a molecule having similarcharacteristics as the polypeptide (e.g., a conservative amino acidsubstitution). For example, certain amino acids can be substituted forother amino acids in a sequence without appreciable loss of activity.Because it is the interactive capacity and nature of a polypeptide thatdefines that polypeptide's biological functional activity, certain aminoacid sequence substitutions can be made in a polypeptide sequence andnevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, andcofactors. It is known in the art that an amino acid can be substitutedby another amino acid having a similar hydropathic index and stillobtain a functionally equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydropathic indices are within +2 ispreferred, those within +1 are particularly preferred, and those within+0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5+1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within+2 is preferred, those within +1 are particularly preferred, and thosewithin +0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide ofinterest.

The term “isolated” is meant to describe a compound of interest (e.g.,nucleic acids) that is in an environment different from that in whichthe compound naturally occurs, e.g., separated from its natural milieusuch as by concentrating a peptide to a concentration at which it is notfound in nature. “Isolated” is meant to include compounds that arewithin samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified. Isolated nucleic acids are at least 60% free,preferably 75% free, and most preferably 90% free from other associatedcomponents.

The term “vector” refers to a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors can beexpression vectors.

The term “expression vector” refers to a vector that includes one ormore expression control sequences

The term “expression control sequence” refers to a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence. Control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and the like. Eukaryotic cells are known to utilizepromoters, polyadenylation signals, and enhancers.

“Transformed,” “transgenic,” “transfected” and “recombinant” refer to ahost organism such as a bacterium or a plant into which a heterologousnucleic acid molecule has been introduced. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof. A“non-transformed,” “non-transgenic,” or “non-recombinant” host refers toa wild-type organism, e.g., a bacterium or plant, which does not containthe heterologous nucleic acid molecule.

The term “endogenous” with regard to a nucleic acid refers to nucleicacids normally present in the host.

The term “heterologous” refers to elements occurring where they are notnormally found. For example, a promoter may be linked to a heterologousnucleic acid sequence, e.g., a sequence that is not normally foundoperably linked to the promoter. When used herein to describe a promoterelement, heterologous means a promoter element that differs from thatnormally found in the native promoter, either in sequence, species, ornumber. For example, a heterologous control element in a promotersequence may be a control/regulatory element of a different promoteradded to enhance promoter control, or an additional control element ofthe same promoter. The term “heterologous” thus can also encompass“exogenous” and “non-native” elements.

The term “percent (%) sequence identity” is defined as the percentage ofnucleotides or amino acids in a candidate sequence that are identicalwith the nucleotides or amino acids in a reference nucleic acidsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

For purposes herein, the % sequence identity of a given nucleotides oramino acids sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given sequence Cthat has or comprises a certain % sequence identity to, with, or againsta given sequence D) is calculated as follows:

100 times the fraction W/Z,

where W is the number of nucleotides or amino acids scored as identicalmatches by the sequence alignment program in that program's alignment ofC and D, and where Z is the total number of nucleotides or amino acidsin D. It will be appreciated that where the length of sequence C is notequal to the length of sequence D, the % sequence identity of C to Dwill not equal the % sequence identity of D to C.

The term “stringent hybridization conditions” as used herein mean thathybridization will generally occur if there is at least 95% andpreferably at least 97% sequence identity between the probe and thetarget sequence. Examples of stringent hybridization conditions areovernight incubation in a solution comprising 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared carrier DNA such as salmon sperm DNA, followed by washing thehybridization support in 0.1×SSC at approximately 65° C. Otherhybridization and wash conditions are well known and are exemplified inSambrook et al, Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor, N.Y. (2000).

As used herein, the term “low stringency” refers to conditions thatpermit a polynucleotide or polypeptide to bind to another substance withlittle or no sequence specificity.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially free(at least 60% free, preferably 75% free, and most preferably 90% free)from other components normally associated with the molecule or compoundin a native environment.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.

Unless otherwise indicated, the disclosure encompasses conventionaltechniques of molecular biology, microbiology, cell biology andrecombinant DNA, which are within the skill of the art. See, e.g.,Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rdedition (2001); Current Protocols In Molecular Biology [(F. M. Ausubel,et al. eds., (1987)]; Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds.(1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.);the series Methods in Enzymology (Academic Press, Inc.): PCR 2: APractical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.(1995)].

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes VII, published by Oxford University Press,2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology,published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.),Molecular Biology and Biotechnology, a Comprehensive Desk Reference,published by VCH Publishers, Inc., 1995; Sambrook and Russell. (2001)Molecular Cloning: A Laboratory Manual 3rd. edition, Cold Spring HarborLaboratory Press.

II. Compositions

A. tRNA

Non-naturally occurring tRNA^(Sec) suitable for carrying selenocysteineand facilitating synthesis of selenopeptides without requiring a SECISin the mRNA encoding the peptide are disclosed. Also disclosed areaminoacylated non-naturally occurring tRNA^(Sec). Using the methodsdiscussed in more detail below, the tRNA^(Sec) disclosed herein arecapable of being aminoacylated to form a Sec-tRNA^(Sec) which canfacilitate insertion of selenocysteine into nascent polypeptide chains.Typically, the non-naturally occurring tRNA^(Sec) (1) can be recognizedby SerRS and by EF-Tu, or variants thereof; and is characterized by oneor more of the following elements: (2) when aminoacylated with serinethe non-naturally occurring Ser-tRNA^(Sec) can be converted tonon-naturally occurring Sec-tRNA^(Sec) by SelA, or variant thereof; (3)when aminoacylated with serine the non-naturally occurringSer-tRNA^(Sec) can be phosphorylated by PSTK or variant thereof; (4)when aminoacylated with phosphorylated serine the non-naturallyoccurring Sep-tRNA^(Sec) can serve as a substrate for SepSecS or variantthereof; and combinations thereof. In some embodiments, thenon-naturally occurring tRNA^(Sec) is characterized by elements (1) and(2). In some embodiments, the non-naturally occurring tRNA^(Sec) ischaracterized by elements (1), (3), and (4). In some embodiments, thenon-naturally occurring tRNA^(Sec) is characterized by elements (1),(2), (3), and (4). Typically, the non-naturally occurring Sec-tRNA^(Sec)can be bound by EF-Tu. The Sec can be incorporated into a growingpeptide chain at a codon of the mRNA that recognizes the anticodon ofthe tRNA^(Sec). Preferably, EF-Tu does not bind Sep-tRNA^(Sec). In someembodiments, EF-Tu is less efficient at incorporating Ser-tRNA^(Sec)than Sec-tRNA^(Sec) into the growing peptide chain.

The non-naturally occurring tRNA^(Sec) do not require a SECIS element inan mRNA to be incorporated into a growing polypeptide chain duringtranslation. Typically the anticodon of the non-naturally occurringtRNA^(Sec) is recognized or hybridizes to a stop codon. Typically thenon-naturally occurring tRNA^(Sec) can facilitate incorporation of a Secinto a growing peptide chain without the activity of SelB.

1. Substrates for EF-Tu

EF-Tu is a prokaryotic elongation factor that mediates the entry of theaminoacyl-tRNA into a free site of the ribosome. Endogenous prokaryotictRNAs, typically include an antideterminant element, which preventsrecognition of a Sec-tRNA^(Sec) by the elongation factor EF-Tu. In someembodiments, the disclosed tRNA can be a substrate for EF-Tu. Therefore,in some embodiments, the disclosed tRNA is a variant of an endogenoustRNA^(Sec) that has been modified to inactivate the antideterminantelement. The antideterminant element can be modified, mutated, ordeleted so that tRNA is an acceptable substrate for EF-Tu. For examplethe antideterminant element in E. coli tRNA^(Sec) is located in the 8th,9th and 10th bp in the acceptor branch of tRNA^(Sec) (encoded by selC),corresponding to the last base pair in the amino acid acceptor stem andthe two first pairs in the T-stem (Rudinger, et al., EMBO J.,15(3):650-57 (1996), and can be referred to as C7•G66/G49•U65/C50•G64according the numbering in Schon, et al., Nucleic Acids Res.,17(18):7159-7165 (1989). Accordingly, in some embodiments, thenon-naturally occurring tRNA^(Sec) is a naturally occurring tRNA^(Sec)where the corresponding antideterminant sequence is mutated or deletedsuch that the non-naturally occurring tRNA^(Sec) is a substrate forEF-Tu.

2. Substrates for PSTK

PSTK is a kinase in archaeal and eukaryotic systems that phosphorylatesSer-tRNA^(Sec) to O-phosphoseryl-tRNA^(Sec), an activated intermediatefor selenocysteine biosynthesis. Accordingly, in some embodiments, onceaminoacylated with serine, the non-naturally occurring tRNA can serve asa substrate for a PSTK, or variant thereof. The enzyme activity of PSTKis strictly tRNA^(Sec)-dependent. PSTK does not hydrolyze ATP in theabsence of tRNA nor in the presence of Ser-tRNA^(Ser). The binding oftRNA^(Ser), however, promotes ATP hydrolysis (R. Lynn Sherrer, et al.,Nucleic Acids Res., 36(4): 1247-1259 (2008)). This indicates thattRNA^(Sec) might play an essential role in positioning the Ser moietyfor initiating phosphoryl transfer. Compared to aminoacyl-tRNAsynthetases, PSTK has approximately 20-fold higher affinity toward itssubstrate, Ser-tRNA^(Sec) (Km=40 nM) (R. Lynn Sherrer, et al., NucleicAcids Res., 36(4): 1247-1259 (2008)), which may compensate for the lowabundance of tRNA^(Sec) in vivo. The concentration of tRNA^(Sec) in vivois at least 10-fold lower than tRNA^(Ser) in tRNA^(Sec)-rich tissuessuch as liver, kidney and testes in rat (Diamond, et al., J. Biol.Chem., 268:14215-14223 (1993)).

The crystal structure of Methanocaldococcus jannaschii PSTK (MjPSTK)places archaeal PSTK identity elements (G2:C71 and the C3:G70) (Sherrer,et al., Nucleic Acids Res, 36:1871-1880 (2008)). within contact of theprotein dimer interface. The second base pair in the acceptor stem ishighly conserved as C2:G71 in eukaryotic tRNA^(Sec), and mutation ofG2:C71 to C2:G71 in archaeal tRNA^(Sec) resulted in a Ser-tRNA^(Sec)variant that is phosphorylated inefficiently (Sherrer, et al., NucleicAcids Res, 36:1871-1880 (2008). The A5-U68 base pair in Methanococcusmaripaludis tRNA^(Ser) has some antideterminant properties for PSTK(Sherrer, et al., NAR, 36(6):1871-1880 (2008)). Moreover, the eukaryoticPSTK has been reported to recognize the unusual D-arm of tRNA^(Sec) asthe major identity element for phosphorylation (Wu and Gross EMBO J.,13:241-248 (1994)). Accordingly, in some embodiments, the disclosedtRNAs include residues in the acceptor stem, the D-arm, or combinationsthereof that are necessary for the tRNA to serve as a substrate for aPSTK.

3. Substrate for SepSecS

The conversion of phosphoseryl-tRNA^(Sec) (Sep-tRNA^(Sec)) toselenocysteinyl-tRNA^(Sec) (Sec-tRNA^(Sec)) is the last step of Secbiosynthesis in both archaea and eukaryotes, and it is catalyzed bytetratmeric O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS).It is believed that one SepSecS homodimer interacts with thesugar-phosphate backbone of both the acceptor-TΨC and the variable armsof tRNA^(Sec), while the other homodimer interacts specifically with thetip of the acceptor arm through interaction between the conserved Arg398and the discriminator base G73 of human tRNA^(Sec).

The co-crystal structure of SepSecS and tRNA^(Sec) also suggests thatthe 9 bp acceptor stem of tRNA^(Sec) is probably important forrecognition by the enzyme (Palioura, S, Sherrer, R L, Steitz, T A, Söll,D & Simonovic, M (2009) Science 325:321-325). According to structuralanalysis, the acceptor-T-variable arm elbow region of tRNA^(Sec)(including bases G50, G51, C64, C65 in the human tRNA^(Sec) that arerecognized by SepSecS) may be critical for recognition by SepSecS.Accordingly, in some embodiments, the disclosed tRNAs include residuesin the acceptor-TΨC, the variable arms of tRNA^(Sec), the tip of theacceptor arm, or combinations thereof necessary for the tRNA to serve asa substrate for SepSecS. In some embodiments, the G50, G51, C64, C65elements of human tRNA^(Sec) are present in the non-naturally occurringtRNA^(Sec).

The SepSecS enzyme itself can also be mutated to engineer enzymevariants that accept a substrate somewhat less ideal than naturallyoccurring tRNA^(Sec). It is believed that His30, Arg33, Lys38 in SepSecSform key interactions with the protomer and G50, U51, C64 and C65 of thetRNA. Therefore, mutation of some of these residues could result in aSepSecS variant that is better able to recognize one of thenon-naturally occurring tRNA^(Sec). The formed Sec-tRNA^(Sec) can bescreened in the formate dehydrogenase-benzyl viologen assay [e.g.,(Yuan, J, Palioura, S, Salazar, J C, Su, D, O'Donoghue, P, Hohn, M J,Cardoso, A M, Whitman, W B & Söll, D (2006), Proc Natl Acad Sci USA103:18923-18927; Palioura, S, Sherrer, R L, Steitz, T A, Söll, D &Simonovic, M (2009) Science 325:321-325)]. Other assays include standardWolfson assay [e.g., (Yuan, J, Palioura, S, Salazar, J C, Su, D,O'Donoghue, P, Hohn, M J, Cardoso, A M, Whitman, W B & Söll, D (2006)Proc Natl Acad Sci USA 103:18923-18927; Palioura, S, Sherrer, R L,Steitz, T A, Söll, D & Simonovic, M (2009) Science 325:321-325)],labeling with [75Se]selenite in the presence of selenophosphate synthase(SelD) [e.g., (Yuan, J, Palioura, S, Salazar, J C, Su, D, O'Donoghue, P,Hohn, M J, Cardoso, A M, Whitman, W B & Söll, D (2006) Proc Natl AcadSci USA 103:18923-18927)], and using [14C] or [3H]serine in the initialcharging reaction.

In some embodiments, a SepCysS is used instead of SepSecS. SepCysS is akey PLP-dependent enzyme in Cys-tRNA formation in methanogens. Itconverts Sep-tRNA^(Cys) into Cys-tRNA^(Cys) using thiophosphate assulfur donor. The enzyme's crystal structure is established (Fukunaga, R& Yokoyama, S (2007) Nat Struct Mol Biol 14:272-279.) and its mechanism(Liu, Y., Dos Santos, P. C., Zhu, X., Orlando, R., Dean, D. R., Söll, D.and Yuan, J. (2012) J. Biol. Chem. 287, 5426-5433) is different fromthat of SepSecS (Palioura, S, Sherrer, R L, Steitz, TA, Söll, D &Simonovic, M (2009) Science 325:321-325.). The length of the acceptorstem of its tRNA substrates is not critical and acceptor helices between7-9 bp are acceptable. Therefore, this enzyme's active site can beengineered to allow selenophosphate (instead of thiophosphate) toparticipate in the reaction.

4. Primary Structure

tRNAs can be described according to their primary structure (i.e., thesequence from 5′ to 3′) as well as their secondary structure. Thesecondary structure of tRNA is typically referred to as a “cloverleaf”,which assumes a 3D L-shaped tertiary structure through coaxial stackingof the helices. FIG. 2 illustrates a typical human tRNA^(Sec), whichincludes an acceptor arm, a D-arm, an anticodon arm, a variable arm, anda TΨC-arm.

In some embodiments the non-naturally occurring tRNA^(Sec) sharessequence identity or sequence homology with a naturally occurring tRNA,for example a naturally occurring tRNA^(Sec), or a naturally occurringtRNA^(Ser).

a. Variants of Naturally Occurring tRNA^(Sec)

The non-naturally occurring tRNA^(Sec) disclosed herein can be a variantof a naturally occurring tRNA^(Sec). The naturally occurring tRNA^(Sec)can be from a prokaryote, including but not limited to E. coli, anarchaea, including, but not limited to, M. maripaludis and M.jannaschii, or a eukaryote including, but not limited to human.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof an E. coli tRNA^(Sec), for example,GGAAGAUCGUCGUCUCCGGUGAGGCGGCUGGACUUCAAAUCCAGUUGGGGCCGCCAGCGGUCCCGGGCAGGUUCGACUCCUGUGAUC UUCCGCCA (SEQ ID NO: 1),which is depicted in FIG. 3 (left panel).

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof an M. maripaludis tRNA^(Sec), for example,

(SEQ ID NO: 2) GGCACGGGGUGCUUAUCUUGGUAGAUGAGGGCGGACUUCAGAUCCGUCGAGUUCCGUUGGAAUUCGGGGUUCGAUUCCCCCCCUGCG CCGCCA.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof a human tRNA^(Sec), for example,GCCCGGAUGAUCCUCAGUGGUCUGGGGUGCAGGCUUCAAACCUGUAGCUGUCUAGGGACAGAGUGGUUCAAUUCCACCUUUCGGGC GCCA (SEQ ID NO:3), which isdepicted in FIG. 2.

A non-naturally occurring tRNA^(Sec) that is a variant of a naturallyoccurring tRNA^(Sec) can have a nucleic acid sequence at least 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% identical to SEQ ID NO: 1, 2, or 3. Preferably the non-naturallyoccurring tRNA^(Sec) that is a variant of a naturally occurringtRNA^(Sec) is characterized by one or more of the following elements:(1) the non-naturally occurring tRNA^(Sec) can be recognized by SerRSand by EF-Tu, or variants thereof; (2) when aminoacylated with serinethe non-naturally occurring Ser-tRNA^(Sec) can be converted tonon-naturally occurring Sec-tRNA^(Sec) by SelA or variant thereof; (3)when aminoacylated with serine the non-naturally occurringSer-tRNA^(Sec) can be phosphorylated by PSTK or variant thereof; (4)when aminoacylated with phosphorylated serine the non-naturallyoccurring Sep-tRNA^(Sec) can serve as a substrate for SepSecS or variantthereof.

b. Variants of Naturally Occurring tRNA^(Ser)

The non-naturally occurring tRNASec disclosed herein can be a variant ofa naturally occurring tRNA^(Ser). The naturally occurring tRNA^(Ser) canbe from a prokaryote, including but not limited to E. coli, an archaea,including, but not limited to, M. maripaludis and M. jannaschii, or aeukaryote including, but not limited to human.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof an E. coli tRNA^(Ser), for example,GGAAGUGUGGCCGAGCGGUUGAAGGCACCGGUCUUGAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGCUUCCGCC A (SEQ ID NO:4), depictedin FIG. 3 (right panel).

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof an M. maripaludis tRNA^(Ser), for example,

(SEQ ID NO: 5) GCAGAGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUGAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCUCUGCG CCA.

A non-naturally occurring tRNA^(Sec) that is a variant of a naturallyoccurring tRNA^(Ser) can have a nucleic acid sequence at least 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more identical to SEQ ID NO:4 or 5. Preferably the non-naturallyoccurring tRNA^(Sec) that is a variant of an E. coli tRNA^(Ser) ischaracterized by one or more of the following elements: (1) thenon-naturally occurring tRNA^(Sec) can be recognized by SerRS and byEF-Tu, or variants thereof; (2) when aminoacylated with serine thenon-naturally occurring Ser-tRNA^(Sec) can be converted to non-naturallyoccurring Sec-tRNA^(Sec) by SelA; (3) when aminoacylated with serine thenon-naturally occurring Ser-tRNA^(Sec) can be phosphorylated by PSTK orvariant thereof; (4) when aminoacylated with phosphorylated serine thenon-naturally occurring Sep-tRNA^(Sec) can serve as a substrate forSepSecS or variant thereof.

c. Chimeric tRNA^(Sec)

The non-naturally occurring tRNA^(Sec) disclosed herein can also be achimeric tRNA including sequences from two or more naturally occurringtRNAs. Some embodiments, the non-naturally occurring tRNA includessequences from a naturally occurring tRNA^(Sec) and a naturallyoccurring tRNA^(Ser). The chimeric tRNA can include nucleic acidsequences or features, for example an antideterminant element, from aprokaryote, including but not limited to E. coli, an archaea, including,but not limited to, M. maripaludis and M. jannaschii, or a eukaryoteincluding, but not limited to, human.

i. E. coli Chimeras

Examples of non-naturally occurring tRNA^(Sec) that are chimeric tRNAsincluding sequence elements from E. coli include, but are not limited toGGAAGAUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCAUCUUCC GCCA (SEQ ID NO:6; E. colitRNA^(UTu)-opal), as depicted in FIG. 3 (center panel);GGAAGAUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCAUCUUCC GCCA (SEQ ID NO:7; E. colitRNA^(UTu)-amber), as depicted in FIG. 3 (center panel); andGGAAGAUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCAUCUUCC GCCA (SEQ ID NO:8; E. colitRNA^(UTu)-ochre).

Other examples of non-naturally occurring tRNA^(Sec) that are chimerictRNAs including sequence elements from E. coli include, but are notlimited to GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGGUGCCGCC A (SEQ ID NO:9; E. colitRNA^(UTu)-opal), as depicted in FIG. 4B;GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGGUGCCGCC A (SEQ ID NO:10; E. colitRNA^(UTu)-amber), as depicted in FIG. 4B; andGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGGUGCCGCC A (SEQ ID NO: 11; E. colitRNA^(UTu)-ochre), which are non-naturally occurring chimeras of E. colitRNA^(Ser) with PSTK identity elements.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof SEQ ID NO:9 such as

(SEQ ID NO: 12; E. coli tRNA^(UTu)-opal - variant 1)GGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGGUGCCC GCCA;(SEQ ID NO: 13; E. coli tRNA^(UTu)-opal - variant 2)GGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCCGGUGCC GCCA;(SEQ ID NO: 14; E. coli tRNA^(UTu)-opal - variant 3)GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCGGUGCCGCC A;(SEQ ID NO: 15; E. coli tRNA^(UTu)-opal - variant 4)GGGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCCGGUGCC CGCCA;(SEQ ID NO: 16; E. coli tRNA^(UTu)-opal - variant 5)GGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCGGUGCCC GCCA;(SEQ ID NO: 17; E. coli tRNA^(UTu)-opal - variant 6)GGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCCGGUGCC GCCA; or(SEQ ID NO: 18; E. coli tRNA^(UTu)-opal - variant 7)GGGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCCGGUGCC CGCCA

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof SEQ ID NO:10 such as

(SEQ ID NO: 19; E. coli tRNA^(UTu)-amber - variant 1)GGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGGUGCCC GCCA;(SEQ ID NO: 20; E. coli tRNA^(UTu)-amber - variant 2)GGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCCGGUGCC GCCA;(SEQ ID NO: 21; E. coli tRNA^(UTu)-amber - variant 3)GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCGGUGCCGCC A;(SEQ ID NO: 22; E. coli tRNA^(UTu)-amber - variant 4)GGGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCCGGUGCC CGCCA;(SEQ ID NO: 23; E. coli tRNA^(UTu)-amber - variant 5)GGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCGGUGCCC GCCA;(SEQ ID NO: 24; E. coli tRNA^(UTu)-amber - variant 6)GGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCCGGUGCC GCCA;(SEQ ID NO: 25; E. coli tRNA^(UTu)-amber - variant 7)GGGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCCGGUGCC CGCCA;

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof SEQ ID NO: 11 such as

(SEQ ID NO: 26; E. coli tRNA^(UTu)-ochre - variant 1)GGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCGGUGCCC GCCA;(SEQ ID NO: 27; E. coli tRNA^(UTu)-ochre - variant 2)GGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCCGGUGCC GCCA;(SEQ ID NO: 28; E. coli tRNA^(UTu)-ochre - variant 3)GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCGGUGCCGCC A;(SEQ ID NO: 29; E. coli tRNA^(UTu)-ochre - variant 4)GGGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCAGAGUUCGAAUCUCUGCCGGUGCC CGCCA;(SEQ ID NO: 30; E. coli tRNA^(UTu)-ochre - variant 5)GGGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCGGUGCCC GCCA;(SEQ ID NO: 31; E. coli tRNA^(UTu)-ochre - variant 6)GGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCCGGUGCC GCCA; or(SEQ ID NO: 32; E. coli tRNA^(UTu)-ochre - variant 7)GGGCACUGGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGGGUUCCUGAGUUCGAAUCUCAGCCGGUGCC CGCCA

A non-naturally occurring tRNA^(Sec) that is a chimeric E. coli tRNA canhave a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical toSEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32. Preferably thenon-naturally occurring tRNA^(Sec) that is a chimeric E. coli tRNA^(Sec)is characterized by one or more of the following elements: (1) thenon-naturally occurring tRNA^(Sec) can be recognized by SerRS and byEF-Tu, or variants thereof; (2) when aminoacylated with serine thenon-naturally occurring Ser-tRNA^(Sec) can be converted to non-naturallyoccurring Sec-tRNA^(Sec) by SelA or variant thereof; (3) whenaminoacylated with serine the non-naturally occurring Ser-tRNA^(Sec) canbe phosphorylated by PSTK or variant thereof; (4) when aminoacylatedwith phosphorylated serine the non-naturally occurring Sep-tRNA^(Sec)can serve as a substrate for SepSecS or variant thereof.

ii. M. maripaludis Chimeras

Examples of non-naturally occurring tRNA^(Sec) that are chimeric tRNAsincluding sequences elements from M. maripaludis include, but are notlimited to,

(SEQ ID NO: 33; M. maripaludis tRNA^(UTu)-opal)GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCGCGCCG CCA,as depicted in FIG. 4A; (SEQ ID NO: 34; M. maripaludis tRNA^(UTu)-amber)GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCGCGCCG CCA,as depicted in FIG. 4A; (SEQ ID NO: 35; M. maripaludis tRNA^(UTu)-ochre)GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCGCGCCG CCA.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof SEQ ID NO:33 such as

(SEQ ID NO: 36; M. maripaludis tRNA^(UTu)-opal -  variant 1)GGGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCGCGCC CGCCA;(SEQ ID NO: 37; M. maripaludis tRNA^(UTu)-opal - variant 2)GGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCCGCGC CGCCA;(SEQ ID NO: 38; M. maripaludis tRNA^(UTu)-opal - variant 3)GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCGCGCCG CCA;(SEQ ID NO: 39; M. maripaludis tRNA^(UTu)-opal - variant 4)GGGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCCGCG CCCGCCA;(SEQ ID NO: 40; M. maripaludis tRNA^(UTu)-opal -  variant 5)GGGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCGCGCC CGCCA;(SEQ ID NO: 41; M. maripaludis tRNA^(UTu)-opal - variant 6)GGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCCGCGC CGCCA; or (SEQ ID NO: 42; M. maripaludis tRNA^(UTu)-opal - variant 7)GGGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCCGCG CCCGCCA.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof SEQ ID NO:34 such as

(SEQ ID NO: 43; M. maripaludis tRNA^(UTu)-amber - variant 1)GGGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCGCGCC CGCCA;(SEQ ID NO: 44; M. maripaludis tRNA^(UTu)-amber - variant 2)GGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCCGCGC CGCCA;(SEQ ID NO: 45; M. maripaludis tRNA^(UTu)-amber - variant 3)GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCGCGCCG CCA;(SEQ ID NO: 46; M. maripaludis tRNA^(UTu)-amber - variant 4)GGGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCCGCG CCCGCCA;(SEQ ID NO: 47; M. maripaludis tRNA^(UTu)-amber - variant 5)GGGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCGCGCC CGCCA;(SEQ ID NO: 48; M. maripaludis tRNA^(UTu)-amber - variant 6)GGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCCGCGC CGCCA; or(SEQ ID NO: 49; M. maripaludis tRNA^(UTu)-amber - variant 7)GGGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCCGCG CCCGCCA.

In some embodiments, the non-naturally occurring tRNA^(Sec) is a variantof SEQ ID NO:35 such as

(SEQ ID NO: 50; M. maripaludis tRNA^(UTu)-ochre - variant 1)GGGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCGCGCC CGCCA;(SEQ ID NO: 51; M. maripaludis tRNA^(UTu)-ochre - variant 2)GGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCCGCGC CGCCA;(SEQ ID NO: 52; M. maripaludis tRNA^(UTu)-ochre - variant 3)GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCGCGCCG CCA;(SEQ ID NO: 53; M. maripaludis tRNA^(UTu)-ochre - variant 4)GGGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGGGGGUUCAAAUCCCUCCCCGCG CCCGCCA;(SEQ ID NO: 54; M. maripaludis tRNA^(UTu)-ochre - variant 5)GGGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCGCGCC CGCCA;(SEQ ID NO: 55; M. maripaludis tRNA^(UTu)-ochre - variant 6)GGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCCGCGC CGCCA; or(SEQ ID NO: 56; M. maripaludis tRNA^(UTu)-ochre - variant 7)GGGCGCGGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUGGGGAGCGUGGGUUCAAAUCCCACCCCGCG CCCGCCA.

A non-naturally occurring tRNA^(Sec) that is a chimeric M. maripaludistRNA can have a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to SEQ ID NO:33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56. Preferably thenon-naturally occurring tRNA^(Sec) that is a chimeric M. maripaludistRNA^(Sec) is characterized by one or more of the following elements:(1) the non-naturally occurring tRNA^(Sec) can be recognized by SerRSand by EF-Tu, or variants thereof; (2) when aminoacylated with serinethe non-naturally occurring Ser-tRNA^(Sec) can be converted tonon-naturally occurring Sec-tRNA^(Sec) by SelA or variant thereof; (3)when aminoacylated with serine the non-naturally occurringSer-tRNA^(Sec) can be phosphorylated by PSTK or variant thereof; (4)when aminoacylated with phosphorylated serine the non-naturallyoccurring Sep-tRNA^(Sec) can serve as a substrate for SepSecS or variantthereof.

5. Secondary Structure

The tRNAs disclosed herein typically include an acceptor arm, a D-arm,an anticodon arm, a variable arm, and a TPC-arm, as described in moredetail below.

a. Acceptor Arm

The non-naturally occurring tRNA^(Sec) disclosed herein includes anacceptor arm. The acceptor arm is the end of a tRNA molecule to which anamino acid becomes bound. It contains both the 5′ and 3′ ends of thetRNA. The 3′-terminal sequence of cytidine-cytidine-adenosine (CCA)overhangs the end, and the terminal A is the site of ‘acceptance’ of theamino acid.

The acceptor stem refers to the 5′ and 3′ sequences to the acceptor armthat form duplex RNA. The acceptor stem can be separate from the CCAoverhang by one or more nucleotides, for example one or more guanine. Insome embodiments, one or more nucleotides that separate the acceptorstem and the overhang are referred to as the discriminator base(s). Forsome tRNAs, the discriminator base preceding the CCA sequence at the 3′end is important for aminoacylation. The discriminator base caninfluence the stability of the base pair of the acceptor arm onto whichit is stacked which can affect the energetic cost of opening the basepair and modulate the structure of the tRNA near the site ofaminoacylation. For some aminoacyl-tRNA synthetases and other proteinsthat interact with tRNA, these factors could be important for specificrecognition and/or formation of the transition state during catalysis(Lee et al., PNAS, 90(15):7149-52 (1993)). In some embodiments, theacceptor stem and the CCA sequence are separated by a single guaninediscriminator base.

The acceptor stem of the non-naturally occurring tRNA^(Sec) disclosedherein typically include 4 to 12, preferably 5 to 11, more preferably 6to 10, most preferably 7 to 9 base pairs of duplex RNA. In someembodiments, the acceptor stem is 7, 8, or 9 base pairs of duplex RNA.

The acceptor stem can be high in G-C content. For example, in someembodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% of the nucleotides of the acceptor stem.

The 5′ and 3′ sequences of the tRNA that form the acceptor stemtypically form a RNA duplex by Waston-Crick base pairing. The 5′ and 3′sequences of the tRNA that form the acceptor stem are typicallysubstantially complementary. Preferably, the 5′ and 3′ sequences of thetRNA that form the acceptor stem bind to or hybridize to each otherunder conditions of high stringency and specificity. In someembodiments, 5′ sequence of the tRNA that forms the acceptor stem is50%, 60%, 70%, 80%, 85%, 90%, 95%, or more complementary to the 3′sequence of the tRNA that forms the acceptor stem. In some embodimentsthe 5′ and 3′ sequences of the tRNA that form the acceptor stem are 100%complementary.

b. D-Arm

The non-naturally occurring tRNA^(Sec) disclosed herein include a D-arm.The D-arm is typically composed of a D stem of duplex RNA and a D loopof non-duplex RNA. The D stem refers to the two segments of the tRNAprimary sequence in the D-arm that form duplex RNA. The D stem of thenon-naturally occurring tRNA^(Sec) typically include 2 to 8, preferably3 to 7, more preferably 4 to 6, base pairs of duplex RNA. In someembodiments, the D stem is 4, 5, or 6 base pairs of duplex RNA.

The D stem can be high in G-C content. For example, in some embodiments,the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ofthe nucleotides of the D stem.

The two segments of the tRNA that form the D stem typically form a RNAduplex by Waston-Crick base pairing. The two segments of the tRNA thatform the D stem are typically substantially complementary. Preferably,the 5′ and 3′ sequences of the tRNA that form the acceptor stem bind toor hybridize to each other under conditions of high stringency andspecificity. In some embodiments, 5′ segment of the tRNA that forms theD stem is between 25% and 50% complementary to the 3′ segment of thetRNA that forms the D stem. In some embodiments the 5′ segment of thetRNA that forms the D stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, or morecomplementary to the 3′ sequence of the tRNA that forms the D stem. Insome embodiments the 5′ and 3′ sequences of the tRNA that form the Dstem are 100% complementary.

The D loop refers to the part of the D-arm that does not form duplexRNA. The D loop's main function is that of recognition. The D loop cancontain the base dihydrouracil. It is widely believed that it will actas a recognition site for aminoacyl-tRNA synthetase, which is an enzymeinvolved in the aminoacylation of the tRNA molecule. The D-loop can havebetween 3 and 15 nucleotides inclusive, preferably between 4 and 12nucleotides inclusive. In some embodiments the D-loop has 4, 5, 6, 7, 8,9, 10, 11, or 12 nucleotides.

c. Anticodon Arm

The non-naturally occurring tRNA^(Sec) disclosed herein include ananticodon arm. The anticodon arm is typically composed of an anticodonstem of duplex RNA and an anticodon loop of non-duplex RNA. Theanticodon stem refers to the two segments of the tRNA primary sequencein the anticodon arm that form duplex RNA. The anticodon stem of thenon-naturally occurring tRNA^(Sec) disclosed herein typically include 2to 8, preferably 3 to 7, more preferably 4 to 6, base pairs of duplexRNA. In some embodiments, the anticodon stem is 4, 5, or 6 base pairs ofduplex RNA.

The anticodon stem can be high in G-C content. For example, in someembodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% of the nucleotides of the anticodon stem.

The two segments of the tRNA that form the anticodon stem typically forma RNA duplex by Waston-Crick base pairing. The two segments of the tRNAthat form the anticodon stem are typically substantially complementary.Preferably, the 5′ and 3′ sequences of the tRNA that form the anticodonstem bind to or hybridize to each other under conditions of highstringency and specificity. In some embodiments the 5′ segment of thetRNA that forms the anticodon stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%,or more complementary to the 3′ sequence of the tRNA that forms theanticodon stem. In some embodiments the 5′ and 3′ sequences of the tRNAthat form the anticodon stem are 100% complementary.

The anticodon loop refers to the part of the anticodon-arm that does notform duplex RNA. The anticodon loop's main function is to present theanticodon sequence which can hybridize to the target codon in the mRNAsequence of interest. The anticodon sequence can be any three nucleotidesequence that binds by complementary base pairing to the target codonsequence in the mRNA of interest. In some embodiments, the anticodonpairs specifically with only one codon. Some anticodon sequences canpair with more than one codon (i.e., wobble base pairing). In someembodiments, the first nucleotide of the anticodon is inosine orpseudouridine, which can hydrogen bond to more than one base in thecorresponding codon position.

In some embodiments, the anticodon hybridizes to a “stop” codon such asUAA, UAG, or UGA, preferably UAG (amber) or UGA (opal). Accordingly, insome embodiments the sequence of the anticodon is UUA, CUA, UCA,preferably CUA (amber) or UCA (opal) (in the 5′ to 3′ direction). Theanticodon loop can have between 5 and 11 nucleotides inclusive,preferably about 7 nucleotides. In some embodiments the anticodon-loophas 5, 7, or 9 nucleotides. Typically, the three nucleotide anticodonsequence is flanked by an equal number of nucleotides both 5′ and 3′ ofthe anticodon sequence within the anticodon loop.

d. Variable Arm

The non-naturally occurring tRNA^(Sec) disclosed herein includes avariable arm. The variable arm is typically composed of a variable stemof duplex RNA and a variable loop of non-duplex RNA. The variable stemrefers to the two segments of the tRNA primary sequence in the variablearm that form duplex RNA. The variable stem of the non-naturallyoccurring tRNA^(Sec) typically includes 2 to 8, preferably 3 to 7, morepreferably 4 to 6, base pairs of duplex RNA. In some embodiments, thevariable stem is 4, 5, or 6 base pairs of duplex RNA. In someembodiments the variable stem has 9, 10, 11, or more base pairs ofduplex RNA.

The variable stem can be high in G-C content. For example, in someembodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% of the nucleotides of the variable stem.

The two segments of the tRNA that form the variable stem typically forma RNA duplex by Waston-Crick base pairing. The two segments of the tRNAthat form the anticodon stem are typically substantially complementary.Preferably, the 5′ and 3′ sequences of the tRNA that form the variablestem bind to or hybridize to each other under conditions of highstringency and specificity. In some embodiments the 5′ segment of thetRNA that forms the variable stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%,or more complementary to the 3′ sequence of the tRNA that forms thevariable stem. In some embodiments the 5′ and 3′ sequences of the tRNAthat form the variable stem are 100% complementary.

The variable loop refers to the part of the variable-arm that does notform duplex RNA. The variable loop can have between 3 and 7 nucleotidesinclusive, preferably between 4 and 6 nucleotides inclusive. In someembodiments the variable loop has 3, 4, 5, 6, or 7 nucleotides.

e. TΨC-Arm

The non-naturally occurring tRNA^(Sec) disclosed herein includes aTΨC-arm (also referred to herein as a T-arm). The T-arm is the region onthe tRNA molecule that acts as a recognition site for the ribosome, andallows a tRNA-ribosome complex to form during the process of proteinbiosynthesis. The T-arm is typically composed of a T stem of duplex RNAand a T loop of non-duplex RNA. The T stem refers to the two segments ofthe tRNA primary sequence in the T-arm that form duplex RNA. The T stemof the non-naturally occurring tRNA^(Sec) typically includes 2 to 8,preferably 3 to 7, more preferably 4 to 6, base pairs of duplex RNA. Insome embodiments, the T stem is 3, 4, or 5 base pairs of duplex RNA.

The T stem can be high in G-C content. For example, in some embodiments,the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ofthe nucleotides of the T stem.

The two segments of the tRNA that form the T stem typically form a RNAduplex by Waston-Crick base pairing. The two segments of the tRNA thatform the T stem are typically substantially complementary. Preferably,the 5′ and 3′ sequences of the tRNA that form the acceptor stem bind toor hybridize to each other under conditions of high stringency andspecificity. In some embodiments, 5′ segment of the tRNA that forms theT stem is equal to or greater than 50% complementary to the 3′ segmentof the tRNA that forms the T stem. In some embodiments the 5′ segment ofthe tRNA that forms the T stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, ormore complementary to the 3′ sequence of the tRNA that forms the T stem.In some embodiments the 5′ and 3′ sequences of the tRNA that form the Tstem are 100% complementary.

The T loop refers to the part of the T-arm that does not form duplexRNA. In some embodiments the T-loop includes thymidine, pseudouridine,residues, or combinations thereof. The T-loop can have between 3 and 15nucleotides inclusive, preferably between 4 and 12 nucleotidesinclusive. In some embodiments the D-loop has 4, 5, 6, 7, 8, 9, 10, 11,or 12 nucleotides.

f. Linker Nucleotides

The five arms of the tRNA can be linked directly, or can be separated byone or more linker or spacer nucleotides to ensure the tRNA assumes theproper secondary structure. For example, the acceptor arm and the D-armcan separated by 0, 1, 2, 3, or more nucleotides; the D-arm and theanticodon arm can be separated by 0, 1, 2, 3, or more nucleotides; theanticodon arm and the variable arm can be separated by 0, 1, 2, 3, ormore nucleotides; the variable arm and the T-arm can be separated by 0,1, 2, 3, or more nucleotides; and the T-arm and the acceptor arm can beseparated by 0, 1, 2, 3, or more nucleotides.

B. mRNA and Polypeptides of Interest

As discussed in more detail below, the non-naturally occurringtRNA^(Sec) disclosed herein can be used in combination with an mRNA tomanufacture selenocysteine containing polypeptides and proteins. ThemRNA does not require, and preferably does not include, a SECIS element.The mRNA, which encodes a polypeptide of interest, includes one or morecodons that is recognized by the anticodon of the Sec-tRNA^(Sec),referred to herein as an “tRNA^(Sec) recognition codon,” such that tRNAcatalyzes the attachment of a selenocysteine amino acid to the growingpolypeptide chain during translation.

For example, if the tRNA^(Sec) recognition codon is a stop codon, suchas UGA, the mRNA will contain at least one UGA codon where aselenocysteine will be added to the growing polypeptide chain duringtranslation. The tRNA^(Sec) recognition codon can be added to orinserted into any mRNA to add a codon encoding selenocysteine at anydesired location in the amino acid sequence. The tRNA^(Sec) recognitioncodon can be substituted for any existing codon in the mRNA sequence sothat any one or more amino acids from a reference polypeptide sequenceis substituted with selenocysteine during translation. For example, asdiscussed in more detail below, in some embodiments, one or more codonsencoding cysteine in a reference sequence are substituted with atRNA^(Sec) recognition sequence so that the one or more cysteines arereplaced with selenocysteine during translation.

Various types of mutagenesis can be used to modify the sequence of anucleic acid encoding the mRNA of interest to generate the tRNA^(Sec)recognition codon. They include but are not limited to site-directed,random point mutagenesis, homologous recombination (DNA shuffling),mutagenesis using uracil containing templates, oligonucleotide-directedmutagenesis, phosphorothioate-modified DNA mutagenesis, and mutagenesisusing gapped duplex DNA or the like. Additional suitable methods includepoint mismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis and double-strand breakrepair.

In some embodiments, the coding sequence, excluding the tRNA^(Sec)recognition site as discussed above, is further altered for optimalexpression (also referred to herein as “codon optimized”) in anexpression system of interest. Methods for modifying coding sequences toachieve optimal expression are known in the art.

C. Isolated Nucleic Acid Molecules

Non-naturally occurring tRNA^(Sec) and nucleic acids encodingnon-naturally occurring tRNA^(Sec) are disclosed. Also disclosed aremRNAs, cDNAs and other nucleic acids encoding proteins of interest thatare engineered such that a tRNA^(Sec), such as the non-naturallyoccurring tRNA^(Sec) disclosed herein, “reads” at least one codon of themRNA during translation of the protein encoded by the mRNA. As usedherein, “isolated nucleic acid” refers to a nucleic acid that isseparated from other nucleic acid molecules that are present in agenome, including nucleic acids that normally flank one or both sides ofthe nucleic acid in the genome. The term “isolated” as used herein withrespect to nucleic acids also includes the combination with anynon-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule or an RNAmolecule, provided one of the nucleic acid sequences normally foundimmediately flanking that DNA molecule in a naturally-occurring genomeis removed or absent. Thus, an isolated nucleic acid includes, withoutlimitation, a DNA molecule or RNA molecule that exists as a separatemolecule independent of other sequences (e.g., a chemically synthesizednucleic acid, or a cDNA, or RNA, or genomic DNA fragment produced by PCRor restriction endonuclease treatment), as well as recombinant DNA thatis incorporated into a vector, an autonomously replicating plasmid, avirus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), orinto the genomic DNA of a prokaryote or eukaryote. In addition, anisolated nucleic acid can include an engineered nucleic acid such as arecombinant DNA molecule or RNA molecule that is part of a hybrid orfusion nucleic acid. A nucleic acid existing among hundreds to millionsof other nucleic acids within, for example, a cDNA library or a genomiclibrary, or a gel slice containing a genomic DNA restriction digest, isnot to be considered an isolated nucleic acid.

Nucleic acids encoding the tRNA^(Sec) and mRNA disclosed herein may beoptimized for expression in the expression host of choice. In the caseof nucleic acids encoding expressed polypeptides, codons may besubstituted with alternative codons encoding the same amino acid toaccount for differences in codon usage between the organism from whichthe nucleic acid sequence is derived and the expression host. In thismanner, the nucleic acids may be synthesized using expressionhost-preferred codons.

Nucleic acids can be in sense or antisense orientation, or can becomplementary to a reference sequence, for example, a sequence encodingthe disclosed tRNA^(Sec) and mRNA. Nucleic acids can be DNA, RNA,nucleic acid analogs, or combinations thereof. Nucleic acid analogs canbe modified at the base moiety, sugar moiety, or phosphate backbone.Such modification can improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety caninclude deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidineor 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of thesugar moiety can include modification of the 2′ hydroxyl of the ribosesugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribosephosphate backbone can be modified to produce morpholino nucleic acids,in which each base moiety is linked to a six membered, morpholino ring,or peptide nucleic acids, in which the deoxyphosphate backbone isreplaced by a pseudopeptide backbone and the four bases are retained.See, for example, Summerton and Weller (1997) Antisense Nucleic AcidDrug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem.4:5-23. In addition, the deoxyphosphate backbone can be replaced with,for example, a phosphorothioate or phosphorodithioate backbone, aphosphoroamidite, or an alkyl phosphotriester backbone.

D. Methods for Producing Isolated Nucleic Acid Molecules

Isolated non-naturally occurring tRNA^(Sec), nucleic acids encodingnon-naturally occurring tRNA^(Sec), and nucleic acids encodingpolypeptides manufactured using the non-naturally occurring tRNA^(Sec),are disclosed. Isolated nucleic acid molecules can be produced bystandard techniques, including, without limitation, common molecularcloning and chemical nucleic acid synthesis techniques. For example,polymerase chain reaction (PCR) techniques can be used to obtain anisolated nucleic acid encoding a non-naturally occurring tRNA^(Sec). PCRis a technique in which target nucleic acids are enzymaticallyamplified. Typically, sequence information from the ends of the regionof interest or beyond can be employed to design oligonucleotide primersthat are identical in sequence to opposite strands of the template to beamplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length.General PCR techniques are described, for example in PCR Primer: ALaboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring HarborLaboratory Press, 1995.

When using RNA as a source of template, reverse transcriptase can beused to synthesize a complementary DNA (cDNA) strand. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids. See, for example, Lewis (1992)Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

Isolated nucleic acids can be chemically synthesized, either as a singlenucleic acid molecule or as a series of oligonucleotides (e.g., usingphosphoramidite technology for automated DNA synthesis in the 3′ to 5′direction). For example, one or more pairs of long oligonucleotides(e.g., >100 nucleotides) can be synthesized that contain the desiredsequence, with each pair containing a short segment of complementarity(e.g., about 15 nucleotides) such that a duplex is formed when theoligonucleotide pair is annealed. DNA polymerase can be used to extendthe oligonucleotides, resulting in a single, double-stranded nucleicacid molecule per oligonucleotide pair, which then can be ligated into avector. Isolated nucleic acids can also obtained by mutagenesis. Nucleicacids can be mutated using standard techniques, includingoligonucleotide-directed mutagenesis and/or site-directed mutagenesisthrough PCR. See, Short Protocols in Molecular Biology. Chapter 8, GreenPublishing Associates and John Wiley & Sons, edited by Ausubel et al,1992. Examples of nucleic acid amino acid positions relative to areference sequence that can be modified include those described herein.

E. Vectors and Host Cells

Vectors encoding non-naturally occurring tRNA^(Sec) and polypeptidesmanufactured using the non-naturally occurring tRNA^(Sec) are alsoprovided. Nucleic acids, such as those described above, can be insertedinto vectors for expression in cells. As used herein, a “vector” is areplicon, such as a plasmid, phage, virus or cosmid, into which anotherDNA segment may be inserted so as to bring about the replication of theinserted segment. Vectors can be expression vectors. An “expressionvector” is a vector that includes one or more expression controlsequences, and an “expression control sequence” is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence.

Nucleic acids in vectors can be operably linked to one or moreexpression control sequences. Operably linked means the disclosedsequences are incorporated into a genetic construct so that expressioncontrol sequences effectively control expression of a sequence ofinterest. Examples of expression control sequences include promoters,enhancers, and transcription terminating regions. A promoter is anexpression control sequence composed of a region of a DNA molecule,typically within 100 nucleotides upstream of the point at whichtranscription starts (generally near the initiation site for RNApolymerase II).

A “promoter” as used herein is a DNA regulatory region capable ofinitiating transcription of a gene of interest. Some promoters are“constitutive,” and direct transcription in the absence of regulatoryinfluences. Some promoters are “tissue specific,” and initiatetranscription exclusively or selectively in one or a few tissue types.Some promoters are “inducible,” and achieve gene transcription under theinfluence of an inducer. Induction can occur, e.g., as the result of aphysiologic response, a response to outside signals, or as the result ofartificial manipulation. Some promoters respond to the presence oftetracycline; “rtTA” is a reverse tetracycline controlledtransactivator. Such promoters are well known to those of skill in theart.

To bring a coding sequence under the control of a promoter, it isnecessary to position the translation initiation site of thetranslational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the protein encoded by thecoding sequence.

Likewise, although tRNA^(Sec) sequences do not encode a protein, controlsequence can be operably linked to a sequence encoding a tRNA^(Sec), tocontrol expression of the tRNA^(Sec) in a host cell. Methods ofrecombinant expression of tRNA from vectors is known in the art, see forexample, Ponchon and Dardel, Nature Methods, 4(7):571-6 (2007); Massonand Miller, J. H., Gene, 47:179-183 (1986); Meinnel, et al., NucleicAcids Res., 16:8095-6 (1988); Tisné, et al., RNA, 6:1403-1412 (2000).

F. Host Cells

Host cell including the nucleic acids disclosed herein are alsoprovided. Prokaryotes useful as host cells include, but are not limitedto, gram negative or gram positive organisms such as E. coli or Bacilli.In a prokaryotic host cell, a polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant polypeptide. Promoter sequencescommonly used for recombinant prokaryotic host cell expression vectorsinclude lactamase and the lactose promoter system.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. To construct an expression vector using pBR322, anappropriate promoter and a DNA sequence are inserted into the pBR322vector. Other commercially available vectors include, for example, T7expression vectors from Invitrogen, pET vectors from Novagen and pALTER®vectors and PinPoint® vectors from Promega Corporation.

In some embodiments, the host cells are E. coli. The E. coli strain canbe a selA, selB, selC, deletion strain, or combinations thereof. Forexample, the E. coli can be a selA, selB, and selC deletion strain, or aselB and selC deletion strain. Examples of suitable E. coli strainsinclude, but are not limited to, MH5 and MH6.

Yeasts useful as host cells include, but are not limited to, those fromthe genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces.Yeast vectors will often contain an origin of replication sequence, anautonomously replicating sequence (ARS), a promoter region, sequencesfor polyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, (1980)) or otherglycolytic enzymes (Holland et al., Biochem. 17:4900, (1978)) such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer etal., Gene, 107:285-195 (1991), in Li, et al., Lett Appl Microbiol.40(5):347-52 (2005), Jansen, et al., Gene 344:43-51 (2005) and Daly andHearn, J. Mol. Recognit. 18(2):119-38 (2005). Other suitable promotersand vectors for yeast and yeast transformation protocols are well knownin the art.

In some embodiments, the host cells are eukaryotic cells. For example,mammalian and insect host cell culture systems well known in the art canalso be employed to express non-naturally occurring tRNA^(Sec) and mRNAfor producing proteins or polypeptides containing selenocysteine.Commonly used promoter sequences and enhancer sequences are derived fromPolyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome may beused to provide other genetic elements for expression of a structuralgene sequence in a mammalian host cell, e.g., SV40 origin, early andlate promoter, enhancer, splice, and polyadenylation sites. Viral earlyand late promoters are particularly useful because both are easilyobtained from a viral genome as a fragment which may also contain aviral origin of replication. Exemplary expression vectors for use inmammalian host cells are well known in the art.

III. Methods for Manufacturing Proteins Containing Selenocysteine

A. Expression of Selenocysteine Containing Polypeptides

Generally, the canonical amino acids are charged onto their respectivetRNA by their cognate aminoacyl-tRNA synthetase. The aminoacyl-tRNA isthen delivered by EF-Tu to the ribosome (FIG. 1A). In contrast, theendogenous Sec pathway requires several biosynthetic steps. First,tRNA^(Sec) is misacylated to Ser-tRNA^(Sec) by SerRS. While in bacteriaSer-tRNA^(Sec) is directly converted by SelA to Sec-tRNA^(Sec), archaeaand eukaryotes employ an additional phosphorylation step by PSTK to formSep-tRNA^(Sec), which is then converted by SepSecS to the final productSec-tRNA^(Sec) FIG. 1B. Sec-tRNA^(Sec) is bound by elongation factorSelB and delivered to the ribosome. However, reassignment of the opalcodon UGA to a Sec codon is only achieved if SelB also binds to the mRNASECIS hairpin structure.

The compositions disclosed herein can be used to prepare polypeptidesincluding one or more selenocysteine residues from mRNA that does notcontain an SECIS element. The tRNA^(Sec) disclosed herein is recognizedby SerRS and misacylated to form the intermediate Ser-tRNA^(Sec)Next theSer-tRNA^(Sec) is converted to Sec-tRNA^(Sec) by SelA in prokaryoticsystem or hybrid systems, or PSTK and SepSecS in archaeal, eukaryotic,or hybrid systems. Finally, the Sec-tRNA^(Sec) is delivered to theribosome by EF-Tu, where the anticodon of the Sec-tRNA^(Sec) recognizesthe codon engineered to encode a Sec amino acid, and transfers the Seconto the growing polypeptide chain. Accordingly, the non-naturallyoccurring tRNA^(Sec) disclosed herein are typically recognized by SerRS,or a variant thereof, and when aminoacylated with serine the Ser-tRNAcan (1) be a substrate for SelA or a variant thereof; or (2) be asubstrate for PSTK and when aminoacylated with phosphorylated serine theSep-tRNA can serve as a substrate for SepSecS or a variant thereof, and(3) when aminoacylated, the non-naturally occurring Sec-tRNA^(Sec) isrecognized by EF-Tu.

As discussed in more detail below, recombinant proteins includingselenocysteine can be prepared using in vitro transcription/translationor in vivo expression systems. The system can be of prokaryotic,eukaryotic, or archaeal origin or combinations thereof. For example, thesystem can be hybrid system including selenocysteine biogenesis andtranslation factors from prokaryotic, eukaryotic, archaeal origin, orcombinations thereof. In some embodiments, the system is an in vivoprokaryotic expression including an E. coli strain in which theendogenous genes encoding selB, selC, or selA, selB, selC are deleted ormutated to reduce or eliminate expression of endogenous SelA, SelB, SelCor combinations thereof. The selB, selC, or selA, selB, selC mutantstrains can be engineered to express a non-naturally occurringtRNA^(Sec), as well as a PSTK and a SepSecS. In some embodimentsrecombinant SelA is expressed. The PSTK or SepSecS can of eukaryotic orarchaeal origin, or a variant thereof. For example, in one embodiment,the PSTK is a M. maripaludis PSTK and the SepSecS is a M. jannaschiiSepSecS. In some embodiments, SelA, PSTK and SepSecS are all expressedin the expression system.

In some embodiments selenocysteine biogenesis and translation factorsare mutated to improve their specificity or activity for thenon-naturally occurring tRNA^(Sec). In the recombinant tRNA^(Sec)biosynthetic pathway disclosed herein non-naturally occurring tRNA^(Sec)is first misacylated to Ser-tRNA^(Sec) by SerRS, and subsequentlyconverted to Sec-tRNA^(Sec) by SelA, or PSTK and SepSecS, orcombinations thereof. Accordingly, if the SelA, or PSTK and SepSecS,enzymes are not 100% efficient at converting Ser-tRNA^(Sec) toSec-tRNA^(Sec), the system may incorporate Sec or Ser at the desiredposition. Additionally, in some embodiments, recognition of thenon-naturally occurring Sec-tRNA^(Sec) by EF-Tu, is less efficient thanEF-Tu recognition of other naturally occurring aminoacyl-tRNAs. Mutatingthe EF-Tu, SerRS, SelA, PSTK, SepSecS, or combinations thereof canimprove the efficiency or recognition of the enzyme for thenon-naturally occurring tRNA^(Sec), the non-naturally occurringSec-tRNA^(Sec), or various intermediates thereof. Accordingly, in someembodiment, the EF-Tu, SerRS, SelA, PSTK, SepSecS, or combinationsthereof are variants of a naturally occurring protein.

It is understood that if the tRNA^(Sec) recognition codon of the mRNA ofinterest is one of the three mRNA stop codons (UAG, UAA, or UGA)translation of some of the mRNA of interest will terminate at each ofthe tRNA^(Sec) recognition codons, resulting in a heterogeneous mixtureof full-length and truncated proteins. Therefore, in some embodiments,the selenocysteine containing protein is expressed in a system that hasbeen modified or mutated to reduce or eliminate expression of one ormore translation release factors. A release factor is a protein thatallows for the termination of translation by recognizing the terminationcodon or stop codon in an mRNA sequence. Prokaryotic release factorsinclude RF1, RF2 and RF3; and eukaryotic release factors include eRF1and eRF3.

Deletion of one or more release factors may result in “read-through” ofthe intended stop codon. Accordingly, some of recombinant proteinsexpressed in a system with one or more release factors may include oneor more additional amino acids at the C-terminal end of the protein.

The protein of interest can be purified from the truncated proteins andother contaminants using standard methods of protein purification asdiscussed in more detail below.

1) In vitro Transcription/Translation

In one embodiment, the genes encoding a non-naturally occurringtRNA^(Sec), mRNA encoding the protein of interest, mRNA encoding EF-Tu,SerRS, SelA, PSTK, SepSecS, or combinations thereof are synthesized invitro prior to or along with transcription and translation of theprotein of interest. The synthesis of protein from a DNA sequence invitro takes two steps. The first is transcription of an RNA copy and thesecond is the translation of a protein.

In vitro protein synthesis does not depend on having a polyadenylatedRNA, but if having a poly(A) tail is essential for some other purpose avector may be used that has a stretch of about 100 A residuesincorporated into the polylinker region. That way, the poly(A) tail is“built in” by the synthetic method.

Eukaryotic ribosomes read RNAs more efficiently if they have a 5′ methylguanosine cap. RNA caps can be incorporated by initiation oftranscription using a capped base analogue, or adding a cap in aseparate in vitro reaction post-transcriptionally.

The use of in vitro translation systems can have advantages over in vivogene expression when the over-expressed product is toxic to the hostcell, when the product is insoluble or forms inclusion bodies, or whenthe protein undergoes rapid proteolytic degradation by intracellularproteases. Various approaches to in vitro protein synthesis are known inthe art and include translation of purified RNA, as well as “linked” and“coupled” transcription:translation. In vitro translation systems can beeukaryotic or prokaryotic cell-free systems.

Combined transcription/translation systems are available, in which bothphage RNA polymerases (such as T7 or SP6) and eukaryotic ribosomes arepresent. One example of a kit is the TNT® system from PromegaCorporation.

Other suitable in vitro transcription/translation systems include, butare not limited to, the rabbit reticulocyte system, the E. coli S-30transcription-translation system, and the wheat germ based translationalsystem.

2) In Vivo Methods Transcription/Translation

a. Extrachromosomal Expression

Host cells can be genetically engineered (e.g., transformed, transducedor transfected) with the vectors encoding non-naturally occurringtRNA^(Sec) and a nucleic acid encoding the protein of interest, whichcan be, for example, a cloning vector or an expression vector. Thevector can be, for example, in the form of a plasmid, a bacterium, avirus, a naked polynucleotide, or a conjugated polynucleotide. Thevectors are introduced into cells and/or microorganisms by standardmethods including electroporation (From et al., Proc. Natl. Acad. Sci.USA 82, 5824 (1985), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)). Methods of expressing recombinant proteins invarious recombinant expression systems including bacteria, yeast,insect, and mammalian cells are known in the art, see for exampleCurrent Protocols in Protein Science (Print ISSN: 1934-3655 Online ISSN:1934-3663, Last updated January 2012).

Kits are commercially available for the purification of plasmids frombacteria, (see, e.g., GFX™ Micro Plasmid Prep Kit from GE Healthcare;Strataprep® Plasmid Miniprep Kit and StrataPrep® EF Plasmid Midiprep Kitfrom Stratagene; GenElute™ HP Plasmid Midiprep and Maxiprep Kits fromSigma-Aldrich, and, Qiagen plasmid prep kits and QIAfilter™ kits fromQiagen). The isolated and purified plasmids are then further manipulatedto produce other plasmids, used to transfect cells or incorporated intorelated vectors to infect organisms. Typical vectors containtranscription and translation terminators, transcription and translationinitiation sequences, and promoters useful for regulation of theexpression of the particular target nucleic acid. The vectors optionallycomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems.

Useful prokaryotic and eukaryotic systems for expressing and producingpolypeptides are well known in the art include, for example, Escherichiacoli strains such as BL-21, and cultured mammalian cells such as CHOcells.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express non-naturally occurring tRNA^(Sec) and mRNA forproducing proteins or polypeptides containing selenocysteine. Viralbased expression systems are well known in the art and include, but arenot limited to, baculoviral, SV40, retroviral, or vaccinia based viralvectors.

Mammalian cell lines that stably express tRNA and proteins can beproduced using expression vectors with appropriate control elements anda selectable marker. For example, the eukaryotic expression vectorspCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al.(1985) Science 228:810-815) are suitable for expression of recombinantproteins in, for example, Chinese hamster ovary (CHO) cells, COS-1cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCKcells, and human vascular endothelial cells (HUVEC). Additional suitableexpression systems include the GS Gene Expression System™ availablethrough Lonza Group Ltd.

Following introduction of an expression vector by electroporation,lipofection, calcium phosphate, or calcium chloride co-precipitation,DEAE dextran, or other suitable transfection method, stable cell linescan be selected (e.g., by metabolic selection, or antibiotic resistanceto G418, kanamycin, or hygromycin or by metabolic selection using theGlutamine Synthetase-NSO system). The transfected cells can be culturedsuch that the polypeptide of interest is expressed, and the polypeptidecan be recovered from, for example, the cell culture supernatant or fromlysed cells.

b. Expression by Genomic Integration

Methods of engineering a microorganism or cell line to incorporate anucleic acid sequence into its genome are known in the art. For example,cloning vectors expressing a transposase and containing a nucleic acidsequence of interest between inverted repeats transposable by thetransposase can be used to clone the stably insert the gene of interestinto a bacterial genome (Barry, Gene, 71:75-84 (1980)). Stably insertioncan be obtained using elements derived from transposons including, butnot limited to Tn7 (Drahos, et al., Bio/Tech. 4:439-444 (1986)), Tn9(Joseph-Liauzun, et al., Gene, 85:83-89 (1989)), Tn10 (Way, et al.,Gene, 32:369-379 (1984)), and Tn5 (Berg, In Mobile DNA. (Berg, et al.,Ed.), pp. 185-210 and 879-926. Washington, D.C. (1989)). Additionalmethods for inserting heterologous nucleic acid sequences in E. coli andother gram-negative bacteria include use of specialized lambda phagecloning vectors that can exist stably in the lysogenic state (Silhavy,et al., Experiments with gene fusions, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1984)), homologous recombination (Raibaud, etal., Gene, 29:231-241 (1984)), and transposition (Grinter, et al., Gene,21:133-143 (1983), and Herrero, et al., J. Bacteriology,172(11):6557-6567 (1990)).

Methods of engineering other microorganisms or cell lines to incorporatea nucleic acid sequence into its genome are also known in the art. Forexample, integrative plasmids can be used to incorporate nucleic acidssequences into yeast chromosomes. See for example, Taxis and Knop,Bio/Tech., 40(1):73-78 (2006), and Hoslot and Gaillardin, MolecularBiology and Genetic Engineering of Yeasts. CRC Press, Inc. Boca Raton,Fla. (1992). Methods of incorporating nucleic acid sequence into thegenomes of mammalian lines are also well known in the art using, forexample, engineered retroviruses such lentiviruses.

B. Purification of Selenocysteine Containing Polypeptides

Selenocysteine containing polypeptides can be isolated using, forexample, chromatographic methods such as affinity chromatography, ionexhange chromatography, hydrophobic interaction chromatography, DEAE ionexchange, gel filtration, and hydroxylapatite chromatography. In someembodiments, selenocysteine containing polypeptides can be engineered tocontain an additional domain containing amino acid sequence that allowsthe polypeptides to be captured onto an affinity matrix. For example, anFc-containing polypeptide in a cell culture supematant or a cytoplasmicextract can be isolated using a protein A column. In addition, a tagsuch as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can beused to aid polypeptide purification. Such tags can be inserted anywherewithin the polypeptide, including at either the carboxyl or aminoterminus. Other fusions that can be useful include enzymes that aid inthe detection of the polypeptide, such as alkaline phosphatase.Immunoaffinity chromatography also can be used to purify selenocysteinecontaining polypeptides. Selenocysteine containing polypeptides canadditionally be engineered to contain a secretory signal (if there isnot a secretory signal already present) that causes the protein to besecreted by the cells in which it is produced. The secreted proteins canthen conveniently be isolated from the cell media.

In some embodiments, selenocysteine containing polypeptides are isolatedusing activated thiol SEPHAROSE®, for example, Activated ThiolSEPHAROSE® 4B. As discussed above, in the recombinant tRNA^(Sec)biosynthetic pathway disclosed herein non-naturally occurring tRNA^(Sec)is first misacylated to a non-naturally occurring Ser-tRNA^(Sec) bySerRS, and subsequently converted to Sec-tRNA^(Sec) by SelA, or PSTK andSepSecS, or combinations thereof. Accordingly, if the SelA, or PSTK andSepSecS, enzymes are not 100% efficient at converting Ser-tRNA^(Sec) toSec-tRNA^(Sec) the system may incorporate Sec or Ser at the desiredposition, leading to a heterogeneous mixture of proteins. Activatedthiol SEPHAROSE® can be incorporated into the protein purificationprocess to purify Sec containing proteins from the Ser containingcontaminants.

IV. Methods of Using Selenocysteine Containing Polypeptide

The compositions and methods disclosed herein can be used to manufacturepolypeptides and proteins with one or more selenocysteine residues. Insome embodiments, the mRNA encodes a polypeptide that is a naturallyoccurring selenocysteine containing polypeptide. In some embodiments,the mRNA encodes a polypeptide that is not a naturally occurringselenocysteine containing polypeptide. A nucleic acid sequence caninclude a codon that is recognized by the anticodon of a tRNA^(Sec)disclosed herein, for example a nucleic acid encoding a naturallyoccurring selenocysteine containing protein, or can be modified toinclude a codon recognized by the anticodon of a tRNA^(Sec). The nucleicacid sequence encoding the polypeptide can also be codon optimized forexpression in the desired recombinant expression system. The nucleicacid can be expressed from a vector or incorporated into the genome ofthe desired expression system.

A. Recombinant Selenocysteine Containing Peptides—Naturally Occurring

The disclosed compositions and methods can be used for recombinantexpression of naturally occurring selenocysteine containing peptides, orvariants thereof. Selenoproteins exist in all major forms of life,including, eukaryotes, bacteria and archaea. Accordingly, in someembodiments, the mRNA of interest is an mRNA encoding a selenocysteinecontaining peptide from an eukaryote, a bacteria, or an archaea. Thehuman genome encodes at least 25 naturally occurring selenocysteinecontaining peptides (Kryukov, et al, Science, 300:1439-1443 (2003)).Therefore, in some embodiments the mRNA encodes a iodothyroninedeiodinase such as DIO1, DIO02, DIO3; a glutathione peroxidase such asGPX1, GPX2, GPX3, GPX4, or GPX6; a selenoprotein such as SelH, SelI,SelK, SelM, SelN, SelO, SelP, SelR, SelS, SelT, SelV, SelW, or Sel15;selenophosphate synthetase 2 (SPS2); or a thioredoxin reductase such asTXNRD1, TXNRD2, or TXNRD3.

Conditions to be Treated

In some embodiments, recombinant selenocysteine containing polypeptidesprepared according to the claimed methods are administered to a subjectin an effective amount to treat a disease, or one or more symptomsthereof. As discussed in Riaz and Mehmood, JPMI, 26(02):120-133 (2012)and Tapiero, et al., Biomedicine & Pharmacotherapy 57:134-144 (2003),many health effects of low selenium are thought to be due to lack of oneor more specific selenocysteine containing proteins. For example,reduction or loss of one or more selenocysteine containing protein in asubject can be associated with increased oxidative stress in thesubject. Accordingly, a recombinant selenocysteine containing proteincan be administered to subject in an effective amount to increaseantioxidant activity, or reduce oxidative stress in the subject. In someembodiments, the recombinant selenocysteine containing protein can beused to treat or prevent an age-related disorder, asthma, diabetes, aninfectious disease, a cardiovascular disorder, a cancer, maleinfertility, pre-eclampsia, a gastrointestinal disorder, thyroidmetabolism, or another diseases or condition associated with reducedlevels or activity of selenocysteine containing proteins.

B. Recombinant Selenocysteine Containing Peptides—Non-NaturallyOccurring

The disclosed compositions and methods can also be used for producing byrecombinant expression a selenocysteine containing polypeptide variantof any polypeptide that does not naturally contain selenocysteine.

1. Insertion of Selenocysteine

One or more selenocysteines can be added to the beginning, end, and/orinserted into a polypeptide that does not typically have aselenocysteine. Adding one or more selenocysteines can change thebiochemical and functional properties of the protein, for example,change the redox potential of the protein, increase the half-life of theprotein, increase the stability or resistance to degradation, increasethe activity of the protein (such as enzymatic activity), alter thepharmacokinetics of the protein, alter the binding affinity (such as thebinding affinity of an antibody to antigen or ligand to receptor),change the folding properties of the protein, induce new epitopes ontothe protein, or tag the protein for purification.

In some embodiments, the one or more selenocysteines changes thebiochemical properties of the protein so it can be easily purified afterrecombinant expression. In some embodiments, selenocysteine can be addedto a protein and used as a purification tag. For example, activatedthiol SEPHAROSE®, or an equivalent thereof, can be incorporated into theprotein purification process to purify Sec containing proteins fromcontaminants.

2. Substitution with Selenocysteine

In some embodiments, selenocysteine is substitute for one or morenaturally occurring cysteines.

Reversible oxidation of thiols to disulfides or sulfenic acid residuescontrols biological functions in at least three general ways, bychemically altering active site cysteines, by altering macromolecularinteractions, and by regulating activity through modification ofallosteric Cys (reviewed in Jones, Am. J. Physiol., 295(4):C849-868(2008)). Half of all enzyme activities are sensitive to eitheroxidation, reaction with electrophiles, or interaction with metal ions.Enzymes with active-site Cys include caspases, kinases, phosphatases,and proteases. Cys is also a component of active sites of iron-sulfurclusters of electron transfer proteins and a element of zinc fingers intranscription factors and zinc-binding domains of metallothioneins. Cysresidues are also conserved in structural proteins such as actin anddocking proteins such as 14-3-3. Oxidation of Cys residues in αIIbβ3integrin controls platelet activation. Cys-rich regions are present inplasma membrane receptors and ion channels, including the NMDAreceptors, EGF receptor, and others. Thus reversible oxidation of activesite thiols can provide a common and central “on-off” mechanism forcontrol of cell functions.

β-Actin contains a conserved Cys, which results in reversible binding ofproteins, S-GS-ylation, and crosslinking of actin filaments uponoxidation. Oxidation functions in glucocorticoid receptor translocationinto nuclei, and oxidation controls export of yeast AP-1 (Yap-1) fromnuclei. Disulfide crosslinks control fluidity of mucus. Such changes inprotein structure and interaction due to reversible oxidation canprovide a central mechanism for specificity in redox signaling. Inaddition to containing active site and/or structural thiols, manyproteins contain Cys which regulate activity by an allosteric mechanism.This type of regulation can provide a “rheostat” rather than an “on-off”switch, thereby providing a means to throttle processes by GS-ylation orS-nitrosylation.

Many naturally occurring selenoproteins with known functions areoxidoreductases which contain catalytic redox-active Sec (Jacob C, etal., Angew. Chem. Int. Ed. Engl., 42:4742-4758 (2003)). Variants of thenaturally occurring selenoprotein in which the Sec residues are replacedwith Cys residues are typically 100-1,000 times less active (JohanssonL, et al, Biochim. Biophys. Acta., 1726:1-13 (2005)). Furthermore,analogs of naturally occurring proteins where one or more Cys residuesare replaced with Sec can generate analogs that retain the folding ofthe native peptides, are more potent, and have the same or greaterbiological activity (Raffa, Life Sci., 87(15-16):451-6 (2010)).

Therefore, in some embodiments, the disclosed compositions and methodsare used to manufacture recombinant variants or analogs where one ormore naturally occurring Cys residues, for example Cys residues in theactive site of an enzyme, are replaced with Sec residues. The methodsand compositions can be used to generate analogs that retain a foldingof the protein similar or the same as the native peptides, but are morepotent while having the same or greater biological activity.Substituting one or more naturally occurring Cys residues with a Sec canincrease the activity of the protein by 2, 5, 10, 100, 250, 500, 1,000or more-fold over the activity of the protein that does not contain theSec residue(s). Accordingly, the analogs can be used in therapeutic orresearch applications at a lower dosage, less frequently, with reducedtoxicity, or combinations thereof relative to the naturally occurringprotein.

In some embodiments, the disclosed compositions and methods can be usedto prepare recombinant polypeptides where one or more cysteines thatcontributes to the formation of a disulfide bond in the protein isreplaced with selenocysteine. Therefore, recombinant proteins having oneor more Sec-Sec (diselenide) or Cys-Sec (selenocysteine-cysteine) bondsare disclosed.

A disulfide bond is a covalent bond, usually derived by the coupling oftwo thiol groups. Disulfide bonds in proteins are formed between thethiol groups of cysteine residues. A disulfide bond can stabilize thefolded form of a protein in several ways. For example a disulfide bondcan hold two portions of the protein together, favoring a foldedtopology and contributing to the formation and stability of secondaryand tertiary structures. A disulfide bond can also form the center of ahydrophobic core in a folded protein, i.e., local hydrophobic residuesmay condense around the disulfide bond and onto each other throughhydrophobic interactions. In some cases the hydrophobic core is anenzyme's active site, and the disulfide bond is necessary for enzymaticefficiency or activity.

A diselenide bond, which is formed between two selenocysteine residues,or a selenocysteine-cysteine bond between a selenocysteine and cysteinecan impart similar structural and functional characteristics to theprotein as a disulfide bond. Diselenide and selenocysteine-cysteinebonds are infrequent in nature, but have been reported to be in theactive site of some enzymes, for example the selenocysteine protein SelL(Shchedrina, et al., PNAS, 104(35):13919-13924 (2007)). Diselenide bondshave very low redox potential, but in some cases can be reduced bythioredoxin.

Therefore, in some embodiments, the disclosed compositions and methodsare used to manufacture recombinant variants where one or more naturallyoccurring disulfide bonds are replaced with a diselenide or aselenocysteine-cysteine bond.

Replacing disulfide bonds with diselenide or selenocysteine-cysteinebonds can be used to reduce the redox potential of the bond, increasethe half-life of the protein, increase the activity of the protein,alter the pharmacokinetics of the protein, for example, increase ordecrease the association or dissociation constant, alter the folding andunfolding properties of the protein, or combinations thereof. Forexample, substituting one or more naturally occurring Cys residues witha Sec can increase the activity of the protein by 2, 5, 10, 100, 250,500, 1,000 or more-fold over the activity of the protein that does notcontain the Sec residue(s). Accordingly, the analogs can be used intherapeutic or research applications at a lower dosage, less frequently,with reduced toxicity, or combinations thereof relative to the naturallyoccurring protein.

Exemplary proteins where a naturally occurring Cys can be replaced withSec according to the compositions and methods disclosed herein include,but are not limited to, caspases, kinases, phosphatases, proteases,transcription factors, metallothioneins, structural proteins such asactin and docking proteins such as 14-3-3, integrins such as αIIbβ3,plasma membrane receptors, ion channels, including the NMDA receptors,EGF receptor, and others.

The disclosed compositions and methods can be particularly useful forpreparing recombinant antibodies, antigen binding fragments thereof,fusion proteins including a least one antibody domain (i.e., Ig fusionproteins) with altered properties, and receptor such as T cell receptorsor receptor fragments including the binding domains. Antibodies containinter-chain disulfide bonds which link the heavy and light chains,disulfide bonds that link two heavy chains, and disulfide bonds thatlink the two hinge regions. Antibodies also have disulfide bonds withinthe chains themselves (referred to as intra-chain disulfide bonds). Thedisclosed compositions and methods can be used to prepare recombinantantibodies where one or more disulfide bonds are replaced withdiselenide bonds. The one or more of the inter-chain disulfide bondswhich link the heavy and light chains, the disulfide bonds that link twoheavy chains, the disulfide bonds that link the two hinge regions, theintra-chain disulfide bonds, or combinations thereof can be replacedwith diselenide bonds.

Disulfide bonds in antibodies are important for assembly, stability anddimerization of the antibody. For example, disulfide bonds play acritical role in the stabilization of the immunoglobulin β-sandwich.Under reducing conditions, such as those characteristic of recombinantprotein expression systems, disulfide bonds do not normally form and asa result most antibodies expressed in that compartment are misfolded orinactive (Seo, et al., Protein Sci., 18(2): 259-267 (2009)).Furthermore, stability and homogeneity of therapeutic antibodies areimportant for safety and efficacy of therapeutic antibodies (McAuley, etal, Protein Sci., 17(1): 95-106 (2008)). Undesired biochemical,structural, and conformational forms, such as those generated whendisulfide bonds are reduced, can lead to loss of efficacy and risk ofadverse side effects.

Replacing one or more of the disulfide bonds of an antibody withdiselenide or selenocysteine-cysteine bonds according to the disclosedcompositions and methods can improve the yield, purity, or combinationsthereof, of recombinantly produced antibodies. Replacing one or more ofthe disulfide bonds of an antibody with diselenide orselenocysteine-cysteine bonds according to the disclosed compositionsand methods can also improve stability, increase efficacy, increasehalf-life, reduce toxicity, alter the pharmacokinetics of the antibody,for example, increase or decrease the association or dissociationconstant, or combinations thereof of antibodies, such as therapeuticantibodies.

The antibodies can be xenogeneic, allogeneic, syngeneic, or modifiedforms thereof, such as humanized, single chain or chimeric antibodies.Antibodies may also be anti-idiotypic antibodies specific for a idiotypeof the desired antigen. The term “antibody” is also meant to includeboth intact molecules as well as fragments thereof that include theantigen-binding site and are capable of binding to a desired epitope.These include Fab and F(ab′)₂ fragments which lack the Fc fragment of anintact antibody, and therefore clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nuc. Med. 24:316-325 (1983)). Also included are Fvfragments (Hochman, J. et al., Biochemistry, 12:1130-1135(1973); Sharon,J. et al., Biochemistry, 15:1591-1594 (1976)). These various fragmentscan be produced using conventional techniques such as protease cleavageor chemical cleavage (see, e.g., Rousseaux et al., Meth. Enzymol.,121:663-69 (1986)).

Antibody “formats” and methods of making recombinant antibodies areknown in the art and reviewed in Laffly and Sodoyer, Hum Antibodies,14(1-2):33-35 (2005). Methods of expressing and purifying antibodiesfrom a recombinant expression system are known in the art, see forexample, Knappik and Brundiers, “Recombinant Antibody Expression andPurification,” The Protein Protocols Handbook, Third Edition Edited by:J. M. Walker © Humana Press, a Part of Springer Science+Business Media,LLC (2009).

Therapeutic antibodies that could benefit from replacement of one ormore disulfide bonds with a diselenide or selenocysteine-cysteine bondare known in the art and include, but are not limited to, thosediscussed in Reichert, Mabs, 3(1): 76-99 (2011), for example, AIN-457,bapineuzumab, brentuximab vedotin, briakinumab, dalotuzumab,epratuzumab, farletuzumab, girentuximab (WX-G250), naptumomabestafenatox, necitumumab, obinutuzumab, otelixizumab, pagibaximab,pertuzumab, ramucirumab, REGN88, reslizumab, solanezumab, T1h,teplizumab, trastuzumab emtansine, tremelimumab, vedolizumab,zalutumumab and zanolimumab.

Other therapeutic antibodies that could benefit from replacement of oneor more disulfide bonds with a diselenide bond include antibodiesapproved for use, in clinical trials, or in development for clinical usewhich include, but are not limited to, rituximab (Rituxan®,IDEC/Genentech/Roche) (see for example U.S. Pat. No. 5,736,137), achimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma;HuMax-CD20, an anti-CD20 currently being developed by Genmab, ananti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133(Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM(Intracel), and PRO70769 (PCT/US2003/040426, entitled “ImmunoglobulinVariants and Uses Thereof”), trastuzumab (Herceptin®, Genentech) (seefor example U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibodyapproved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarge),currently being developed by Genentech; an anti-Her2 antibody describedin U.S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Pat. No.4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody in clinicaltrials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883),currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S.Ser. No. 10/172,317), currently being developed by Genmab; 425,EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864;Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al.,1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, ProteinEng. 4(7):773-83); 1CR62 (Institute of Cancer Research) (PCT WO95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993,22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993,67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35;Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YMBiosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat.No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al, 1997,Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institue for CancerResearch, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc NatlAcad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MRI-1 (IVAX,National Cancer Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCTWO 01/88138); alemtuzumab (Campath®, Millenium), a humanized mAbcurrently approved for treatment of B-cell chronic lymphocytic leukemia;muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed byOrtho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), ananti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amcvive®), anti-LFA-3 Fc fusion developed byBiogen), abciximab (ReoPro®), developed by Centocor/Lilly, basiliximab(Simulect®), developed by Novartis, palivizumab (Synagis®), developed byMedimmune, infliximab (Remicade®), an anti-TNFalpha antibody developedby Centocor, adalimumab (Humira®), an anti-TNFalpha antibody developedby Abbott, Humicade®, an anti-TNFalpha antibody developed by Celltech,golimumab (CNTO-148), a fully human TNF antibody developed by Centocor,etanercept (Enbrel®), an p75 TNF receptor Fc fusion developed byImmunex/Amgen, lenercept, an p55TNF receptor Fc fusion previouslydeveloped by Roche, ABX-CBL, an anti-CD 147 antibody being developed byAbgenix, ABX-IL8, an anti-IL8 antibody being developed by Abgenix,ABX-MAI, an anti-MUC18 antibody being developed by Abgenix, Pemtumomab(R1549,90Y-muHMFG1), an anti-MUC1 in development by Antisoma, Therex(R1550), an anti-MUC1 antibody being developed by Antisoma, AngioMab(AS1405), being developed by Antisoma, HuBC-1, being developed byAntisoma, Thioplatin (AS1407) being developed by Antisoma, Antegrene(natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrinantibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin betareceptor (LTBR) antibody being developed by Biogen, CAT-152, ananti-TGF-.beta.2 antibody being developed by Cambridge AntibodyTechnology, ABT 874 (J695), an anti-IL-12 p40 antibody being developedby Abbott, CAT-192, an anti-TGF.beta.1 antibody being developed byCambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxin1antibody being developed by Cambridge Antibody Technology,LyntphoStat-B® an anti-Blys antibody being developed by CambridgeAntibody Technology and Human Genome Sciences Inc., TRAIL-R1mAb, ananti-TRAIL-R1 antibody being developed by Cambridge Antibody Technologyand Human Genome Sciences, Inc. Avastin® bevacizumab, rhuMAb-VEGF), ananti-VEGF antibody being developed by Genentech, an anti-HER receptorfamily antibody being developed by Genentech, Anti-Tissue Factor (ATF),an anti-Tissue Factor antibody being developed by Genentech. Xolair®(Omalizumrnab), an anti-IgE antibody being developed by Genentech,Raptiva® (Efalizumab), an anti-CD11a antibody being developed byGenentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developedby Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-CD4antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Genmab and Amgen, HuMax-Inflam, being developed byGenmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody beingdeveloped by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma,being developed by Genmab and Amgen, HuMax-TAC, being developed byGenmab, IDEC-131, and anti-CD40L antibody being developed by IDECPharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody beingdeveloped by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody beingdeveloped by IDFC Pharmaceuticals, IDEC-152, an anti-CD23 beingdeveloped by IDEC Pharmaceuticals, anti-macrophage migration factor(MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, ananti-idiotypic antibody being developed by Imclone, IMC-1C11, ananti-KDR antibody being developed by Imclone, DC101, an anti-flk-1antibody being developed by Imclone, anti-VE cadherin antibodies beingdeveloped by Imclone, CEA-Cide® (labetuzumab), an anti-carcinoembryonicantigen (CEA) antibody being developed by Immunomedics, LymphoCide®(Epratuzumab), an anti-CD22 antibody being developed by Immunomedics,AFP-Cide, being developed by Immunomedics, MyelomaCide, being developedby Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide,being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody beingdeveloped by Medarex, MDX-060, an anti-CD30 antibody being developed byMedarex, MDX-070 being developed by Medarex, MDX-018 being developed byMedarex, Osidem® (IDM-I), and anti-Her2 antibody being developed byMedarex and Immuno-Designed Molecules, HuMaxe-CD4, an anti-CD4 antibodybeing developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Medarex and Genmab, CNTO 148, an anti-TNFα antibodybeing developed by Medarex and Centocor/J&J. CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAFO, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) ahumanized anti-IgE antibody developed by Genentech and Novartis, andMLN01, an anti-Beta2 integrin antibody being developed by Xoma. Inanother embodiment, the therapeutics include KRN330 (Kirin); huA 33antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha Vintegrins, Centocor); MEDI-522 (alpha V133 integrin, Medimmune);volociximab (αVβ1 integrin, Biogen/PDL); Human mAb 216 (B cellglycosolated epitope, NCI); BiTE MT103 (bispecific CD19×CD3, Medimmune);4G7×H22 (Bispecific Bcell×FcgammaR1, Meclarex/Merck KGa); rM28(Bispecific CD28×MAPG, U.S. Patent No. EP1444268); MDX447 (EMD 82633)(Bispecific CD64×EGFR, Medarex); Catumaxomab (removah) (BispecificEpCAM× anti-CD3, Trion/Fres); Ertumaxomab (bispecific HER2/CD3,Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WXG250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105(CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol MyersSquibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2,Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan)(CD20, Genentech); THIOMAB (Genentech); veltuzumab (hA20) (CD20,Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23,Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDLBiopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30,Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33,Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD 122(CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campathlh(Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-I)(CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293(TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma);ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab(Ticilimumab, CP-675,2) (CTLA4, Pfizer); 1-IGS-ETR1 (Mapatumumab)(DR4TRAIL-R1 agonist, Human Genome Science/Glaxo Smith Kline); AMG-655(DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo);HGS-ETR2 (lexatumumab) (DR5TRAIL-R2 agonist, HGS); Cetuximab (Erbitux)(EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio);Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR,Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab(MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (EpcamGlaxo/Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871(ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307(MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex);Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR betachain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R,Roche); CP 751871 (IGF 1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); B1111022Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6,Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo);Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11(LTβR, Biogen); HuHMFG1 (MUC1, Antisoma/NCI); RAV 12 (N-linkedcarbohydrate epitope, Raven); CAL (parathyroid hormone-related protein(PTH-rP), University of California); CT-011 (PD1, CtireTech); MDX-1106(ono-4538) (PDL Nileclarox/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3(PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591(PSMA, Comell Research Foundation); muJ591 (PSMA, Comell ResearchFoundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab(Remicade) (TNFα, Centocor); A27.15 (transferrin receptor, SalkInstitute, INSERN WO 2005/111082); E2.3 (transferrin receptor, SalkInstitute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF,Tsukuba Research Lab-WO/2000/034337, University of Texas); IMC-18F1(VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone)

In another embodiment, the recombinant protein is a fusion proteinhaving a least one Cys, preferably at least one Cys-Cys bond. In someembodiments, the fusion protein is a fusion protein containing anantibody domain, for example an Ig fusion protein. A fusion proteintypically includes two or more domains, where a first domain including apeptide of interest is fused, directly or indirectly to a secondpolypeptide. In some embodiments, the second domain includes one or moredomains of an Ig heavy chain constant region, preferably having an aminoacid sequence corresponding to the hinge, C_(H2) and C_(H3) regions of ahuman immunoglobulin Cγ1 chain. Construction of immunoglobulin fusionproteins is discussed in Current Protocols in Immunology, (ed. DianeHollenbaugh, Alejandro Aruffo) UNIT 10.19A, Published May 1, 2002, byJohn Wiley and Sons, Inc.

3. Selenocysteine-Containing Polypeptide Conjugates

In some embodiments, the addition of one or more selenocysteines can beused to facilitate linkage of second therapeutic, prophylactic ordiagnostic agent to the selenocysteine containing polypeptide. Methodsof utilizing cysteines as reactive sites for attachment of a secondagent, for example, via a disulfide bridge, are known in the art. Seefor example, Ritter, Pharmaceutical Technology, 42-47 (2012), Miao, etal., Bioconjug. Chem., 19(1):15-19 (2008); and Dosio, et al., Toxins(Basel), 3(7):848-83 (2011). Accordingly, one or more selenocysteinescan be added to a recombinant polypeptide, or substitute for an existingamino acid such as cysteine, to create or replace a reactive site forconjugation of the second agent. The recombinant polypeptide and thesecond agent can be conjugated via a linker. In a preferred embodiment,the recombinant polypeptide engineered to a contain one or moreselenocysteines is an antibody, for example a therapeutic antibody.

In some embodiments, the second agent is a toxin, diagnostic imagingagent, purification ligand or other engineered element that modifies thestability, activity, pharmacokinetics, or other properties of theprotein. The second agent can be a small molecule.

In a preferred embodiment, the second agent is a therapeutic agent. Forexample, the second agent can be a chemotherapeutic drug. The majorityof chemotherapeutic drugs can be divided into: alkylating agents,antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other antitumour agents. All of these drugs affect celldivision or DNA synthesis and function in some way. Additionaltherapeutics include monoclonal antibodies and the new tyrosine kinaseinhibitors e.g. imatinib mesylate (GLEEVEC® or GLIVEC®), which directlytargets a molecular abnormality in certain types of cancer (chronicmyelogenous leukemia, gastrointestinal stromal tumors).

Representative chemotherapeutic agents include, but are not limited to,cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxoland derivatives thereof, irinotecan, topotecan, amsacrine, etoposide,etoposide phosphate, teniposide, epipodophyllotoxins, trastuzumab(HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®),bevacizumab (AVASTIN®), and combinations thereof.

In some preferred embodiments, recombinant antibody including one ormore selenocysteine polypeptides manufactured according to the disclosedmethods is conjugated with second therapeutic agent such as achemotherapeutic drug.

Conditions to be Treated

As discussed above, substituting one or more naturally occurring Cysresidues with a Sec can increase activity, lower dosage, reducetoxicity, improve stability, increase efficacy, increase half-life orcombinations thereof of a selenocysteine containing protein relative toits cysteine containing counterpart. Accordingly, therapeutic proteinscontaining one or more selenocysteine residues can be prepared accordingto the compositions and methods disclosed herein and administered to asubject in need thereof in an effective amount to reduce or alleviateone or more symptoms of a disease or disorder. Therapeutic proteins suchas enzymes and antibodies which contain one or more cysteine residues ordisulfide bonds can be replaced with Sec to increase activity, lowerdosage, reduce toxicity, improve stability, increase efficacy, increasehalf-life, or attach a second agent or combinations thereof arediscussed above and known in the art, and can be administered to subjectto treat diseases or disorders including, but not limited to, infectiousdiseases, cancers, metabolic disorders autoimmune disorders,inflammatory disorders, and age-related disorders.

C. Administration

The recombinant selenocysteine containing polypeptides disclosed hereincan be part of a pharmaceutical composition. The compositions can beadministered in a physiologically acceptable carrier to a host.Preferred methods of administration include systemic or directadministration to a cell. The compositions can be administered to a cellor patient, as is generally known in the art for protein therapyapplications.

The compositions can be combined in admixture with a pharmaceuticallyacceptable carrier vehicle. Therapeutic formulations are prepared forstorage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween®, Pluronics® or PEG.

The compositions can be administered parenterally. As used herein,“parenteral administration” is characterized by administering apharmaceutical composition through a physical breach of a subject'stissue. Parenteral administration includes administering by injection,through a surgical incision, or through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Parenteral formulations can include the active ingredient combined witha pharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Parenteral administration formulationsinclude suspensions, solutions, emulsions in oily or aqueous vehicles,pastes, reconstitutable dry (i.e. powder or granular) formulations, andimplantable sustained-release or biodegradable formulations. Suchformulations may also include one or more additional ingredientsincluding suspending, stabilizing, or dispersing agents. Parenteralformulations may be prepared, packaged, or sold in the form of a sterileinjectable aqueous or oily suspension or solution. Parenteralformulations may also include dispersing agents, wetting agents, orsuspending agents described herein.

Methods for preparing these types of formulations are known. Sterileinjectable formulations may be prepared using non-toxicparenterally-acceptable diluents or solvents, such as water, 1,3-butanediol, Ringer's solution, isotonic sodium chloride solution, and fixedoils such as synthetic monoglycerides or diglycerides. Otherparentally-administrable formulations include microcrystalline forms,liposomal preparations, and biodegradable polymer systems. Compositionsfor sustained release or implantation may include pharmaceuticallyacceptable polymeric or hydrophobic materials such as emulsions, ionexchange resins, sparingly soluble polymers, and sparingly solublesalts.

Pharmaceutical compositions may be prepared, packaged, or sold in abuccal formulation. Such formulations may be in the form of tablets,powders, aerosols, atomized solutions, suspensions, or lozenges madeusing known methods, and may contain from about 0.1% to about 20% (w/w)active ingredient with the balance of the formulation containing anorally dissolvable or degradable composition and/or one or moreadditional ingredients as described herein. Preferably, powdered oraerosolized formulations have an average particle or droplet sizeranging from about 0.1 nanometers to about 200 nanometers whendispersed.

As used herein, “additional ingredients” include one or more of thefollowing: excipients, surface active agents, dispersing agents, inertdiluents, granulating agents, disintegrating agents, binding agents,lubricating agents, sweetening agents, flavoring agents, coloringagents, preservatives, physiologically degradable compositions (e.g.,gelatin), aqueous vehicles, aqueous solvents, oily vehicles and oilysolvents, suspending agents, dispersing agents, wetting agents,emulsifying agents, demulcents, buffers, salts, thickening agents,fillers, emulsifying agents, antioxidants, antibiotics, antifungalagents, stabilizing agents, and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions are known. Suitableadditional ingredients are described in Remington's PharmaceuticalSciences, Mack Publishing Co., Genaro, ed., Easton, Pa. (1985).

Dosages and desired concentrations of the pharmaceutical compositionsdisclosed herein may vary depending on the particular use envisioned.The determination of the appropriate dosage or route of administrationis well within the skill of an ordinary physician. Animal experimentsprovide reliable guidance for the determination of effective doses forhuman therapy. Interspecies scaling of effective doses can be performedfollowing the principles laid down by Mordenti, J. and Chappell, W. “Theuse of interspecies scaling in toxicokinetics” In Toxicokinetics and NewDrug Development, Yacobi et al., Eds., Pergamon Press, New York 1989,pp. 42-96.

EXAMPLES Example 1 Construction of Exemplary Non-Naturally OccurringtRNA^(Sec)

PSTK recognizes certain key bases (called identity elements) in theSep-tRNA^(Sec) substrate. These have been investigated by tRNAmutagenesis and subsequent transplantation of the tRNA^(Sec) identityelements into a tRNA^(Ser) body. The major M. maripaludis tRNA^(Sec)identity elements G2-C71 and C3-G70 were transplanted into M.maripaludis tRNA^(Ser) UGA.

Mutation of the tRNA^(Ser) negative determinant A5-U68 to the C5-G68base pair present in tRNA^(Sec) resulted in a tRNA^(Ser) variant thatphosphorylated with a relative efficiency of 68% as compared towild-type tRNA^(Sec).

Another mutant tRNA with an 8 bp acceptor stem (FIG. 4A) could beconverted to Sep-tRNA^(Sec) with 31% relative efficiency.

Thus, different variants of M. maripaludis tRNA^(Ser) and tRNA^(Sec) areproperly recognized by PSTK.

Since E. coli is a system for Sec incorporation, tRNA^(UTu) candidatesderived from the E. coli tRNA^(Ser) body were also designed (FIGS. 3 and4). E. coli tRNA^(Ser) serves as a major scaffold for tRNA^(UTu), withthe exception of the acceptor stem that originates from E. colitRNA^(Sec) (FIG. 3, center panel, boxed sequence elements). Major EF-Turecognition elements were retained from tRNA^(Ser) as well (FIG. 3,center panel, circled sequence elements). The amber anti-codon CUAconstitutes tRNA^(UTu) _(am) whereas the opal anti-codon UCA constitutestRNA^(UTu) _(op).

The experiments described below utilize the tRNA^(UTu) _(am) or thetRNA^(UTu) _(op), depicted in FIG. 3, center panel (SEQ ID NO:7 and SEQID NO:6 respectively), as indicated.

Example 2 Ser-tRNA^(UTu) _(am) forms Sec-tRNA^(UTu) _(am)

In vitro Ser-tRNA^(UTu) _(am) conversion was confirmed by TLC separationof [³²P] Amp, [³²P] Ser-Amp and [³²P] Sec-Amp recovered by nuclease P1treatment from [α³²P] ATP radiolabeled tRNA^(UTu) _(am), tRNA^(UTu)_(am) after serylation and Ser-tRNA^(UTu) _(am) after incubation in anin vitro Sec formation assay. The TLC was developed for 90 min underacidic conditions using 100 mM ammonium acetate and 5% acetic acid.

Example 3 tRNA^(UTu) Forms the Active Selenoenzyme Formate DehydrogenaseH

Formate Dehydrogenase H (FDH_(H)) is a selenocysteine containing E. colienzyme of known structure (Boyington, et al., Science, 275:1305-08(1997)). A sequence encoding wildtype FDH_(H) (FDH_(Hop)) or anFDH_(H)(FDH_(Ham)) engineered to replace the opal codon with an ambercodon was expressed in e. coli strain MH5 (BW25113 selA selB fdhF).

As shown in FIG. 5, tRNA^(UTu) mediates functional Sec suppression inFDH_(H) E. coli strain MH5 which was complemented with E. coli SelA, M.jannaschii PSTK, and either tRNA^(UTu) _(op) and FDH_(Hop) (1) ortRNA^(UTu) _(am) and FDH_(Ham) (4) and grown anaerobically on LBselective medium supplemented with 0.01 mM IPTG at 30° C. for 24 h.Controls used the same experimental setup with either tRNA^(UT) _(op)(2) or tRNA^(UTu) _(am) (3) omitted and tested the combinations ofFDH_(Hop) and FDH_(Ham) with genomically encoded E. coli tRNA^(Sec) andplasmid encoded selB instead. Cells were overlaid with a top agarcontaining sodium formate and benzyl viologen. FDH_(H) activity was thenassessed by occurrence of a purple coloration upon reduction of benzylviologen.

Example 4 ⁷⁵Se Incorporates into E. coli Selenoprotein FDH_(H)

Cells were grown in the presence of [⁷⁵Se]-selenite in LB mediumsupplemented with 100 M IPTG for protein overexpression.

6×His-tagged FDH_(H) fusion protein was purified and detected bySDS-Page and autoradiography of the gel, with FDH_(H) corresponding tothe protein band at a relative molecular weight of approximately 80,000Da.

Example 5 Sec Complements Thymidylate Synthase a Active Site ResidueCys146

Thymidylate Synthase (ThyA) is an E. coli enzyme with a Cys in theactive site. When the Cys is replaced with a Ser, the protein loses itsenzymatic activity.

As shown in FIG. 6, E. coli MH6 (lacking ThyA) was complemented withexpression constructs encoding either ThyA_(WTCys) (Cys146), ThyA_(Ser)(Cys146Ser) or ThyA_(am) (Cys146Sec) alongside with tRNA^(UTu) _(am) andSelA.

All clones showed growth on LB agar plates while onlyThyA_(am)(Cys146Sec) was able to reconstitute the wild type phenotype(ThyA_(WTCys)) on M9 minimal medium in the absence of thymine.

Example 6 Characterization of Grx1 and GPx1 Mutants

Glutaredoxin-1 (Grx1) is an enzyme with a Cys in the active site, whichis inactive when replaced with a Ser. Constructs were created forexpression of Grx1_(C11am/C14S)Sec (where the Cys 11 codon is replacedwith an amber codon that is recognized by a tRNA^(UTu) _(amber)), aGrx1_(C11S/C14S) (where the Cys 11 codon is replaced with a serinecodon) and Grx1_(C14S) (and wherein Cys 11 codon encodes Cys).

FIG. 7A shows Sec dependent glutathione disulfide oxidoreductaseactivity. Pure Grx1_(C11am/C14S)Sec, Grx1_(C11S/C14S) and Grx1_(C14S)were tested for disulfide oxidoreductase activity. The reduction of amixed disulfide between β-hydroxyethyldisulfide (HED) and glutathione byGrx1 variants is coupled to NADPH consumption by glutathione reductase.Glutathione reductase reconstitutes the reduced Grx1. The reaction wasfollowed at 340 nm at 25° C. as a function of Grx1 concentration.

Pure Grx1_(C11am/C14S)Sec, Grx1_(C11S/C14S) and Grx1_(C14S) were alsotested for peroxidase activity (FIG. 7B). The reduction of tBuOOH to thecorresponding alcohol and water is coupled to the consumption of NADPHby glutathione reductase to reconstitute reduced Grx1. Peroxidaseactivity was determined at 340 nm as a function of reduced glutathioneconcentration at 25° C. Accordingly, peroxidase activity was determinedby monitoring NADPH consumption at 340 nm as a function of theconcentration of reduced glutathione at 25° C.

Peroxidase activity of Sec containing GPx1_(am) and Cys containingGPx1_(Cys) that were overexpressed in E. coli was compared tocommercially available GPx_(hum) from human erythrocytes (FIG. 7C). Theactivity was determined by using the glutathione peroxidase cellularactivity assay kit.

Experiments shown in FIGS. 7A and 7C were performed in triplicates anderror bars indicate the standard error of the mean.

Accordingly, substitution of Cys11 with Sec results in a protein withenzymatic activity, and in some assays, enhanced activity, whilesubstitution of the Cys11 with serine results in a non-functionalprotein.

Sec incorporation was confirmed by mass spectroscopy (FIG. 8). Thepresence of selenocysteine at amino acid position 11 inGrx1_(C11am/C14S)Sec was confirmed by mass spectroscopy. Shown is theMS/MS spectrum of the trypsin-digested Sec-containing fragmentS₉G₁₀U₁₁P₁₂Y₁₃S₁₄V₁₅R₆. Fragments observed in the second massspectrometric analysis of this peptide are labeled with b3, y2, y3, y4and y5. The Sec residue of the peptide in the MS/MS experiment was firsttreated with DTT, and then alkylated with iodoacetamide for oxidativeprotection of the selenol. The unit m/z describes the mass-to-chargeratio.

FIG. 9 is a spectrogram showing the results of Fourier transform ioncyclotron resonance (FT-ICR) mass spectrometry of Grx1_(C11S/C14S)calculated: 10,650 Da. found 10,651 Da.

Sec incorporation was determined spectroscopically by assaying purifiedGrx1_(C11amC14S) with DTNB (Ellman's reagent). Grx1_(C11C/C14S) andGrx1_(C11S/C14S) served as positive and negative controls respectively.

tRNA^(UTu) _(amber) mediated Sec suppression in response to the Ambercodon resulted in a mixture of Sec and Ser containing Grx1_(C11am/C14S)species. From this mixture the Grx1_(C11am/C14S)Sec species wasspecifically recovered by an affinity chromatographic purification usingActivated Thiol Sepharose™ 4B which selectively binds to selenol (thiol)moieties of Sec (Cys) but not to hydroxyl groups of Ser residues.

An overall of 2 mg of the Sec and Ser containing Grx1_(C11am/C14S)mixture were desalted and loaded on an Activated Thiol Sepharose™ 4Bgravity flow chromatography column and incubated with the resin (2 mlbed volume) for 30 min. The column was then washed with lysis buffer (50mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.0) to remove unboundGrx1_(C11am/C14S)Ser protein fraction. Subsequently, immobilizedGrx1_(C11am/C14S)Sec proteins were eluted in the presence of 50 mMreduced glutathione in phosphate buffer. Overall 1.05 mgGrx1_(C11am/C14S)Sec was recovered from the Activated Thiol Sepharose™4B. This represents 52% of the overall Grx1 protein initially subjectedto the Activated Thiol Sepharose™ 4B affinity chromatography.Homogeneity of the recovered Grx1_(C11am/C14S)Sec was accessed by massspectrometry.

Grx1_(C11amC14S) was recovered after affinity chromatography onActivated Thiol SEPHAROSE®. The peaks corresponded to the masses of aGrx1_(C11amC14S) glutathione adduct (11,019.38), a glutathione plus K⁺adduct (11,057.34) and a glutathione plus Met adduct (11,150.42). Theunit m/z describes the mass-to-charge ratio.

FIGS. 11A and 11B shows SerRS kinetics for tRNA^(Sec), tRNA^(Ser) andtRNA^(UTu) _(amber). Varying concentrations (1-30 μM) of tRNA^(Ser),tRNA^(Sec) and tRNA^(UTu) _(amber) were incubated in the presence 30 μML-[14C] serine, 500 nM E. coli SerRS, 5 mM ATP at 37° C. Samples weretaken in the linear range of the aminoacylation reaction and analyzed byscintillation counting. Kinetic parameters were determined byMichaelis-Menten plots of the initial aminoacylation velocity versussubstrate concentration. Each, tRNA^(Sec) tRNA^(Ser) and tRNA^(UTu)_(amber) showed similar kinetic parameters for aminoacylation withserine by SerRS. KM values were determined in the low μM-range around 5μM and Kcat and Kcat/KM at approximately 0.015 s⁻¹ and 0.0025 s⁻¹ μM⁻¹,respectively (FIG. 11A). Ser-tRNA^(UTu)amber is a substrate for SelA invitro. While Ser-tRNA^(Sec) is nearly completely converted toSec-tRNA^(Sec) by SelA, 50% conversion to Sec-tRNA^(UTu) _(amber) isobserved over a course of 20 minutes (FIG. 11B)

FIG. 12 shows in vitro conversion of tRNA^(Sec) and tRNA^(UTu) _(amber)by SelA. 5 μM SelD, 1 mM Na₂SeO₃ and 5 mM ATP were pre-incubated at pH7.2 under anaerobic conditions at 37° C. for 30 min and thensupplemented by 1 μM SelA and 10 μM of [α³²-P] radiolabledSer-tRNA^(Sec) and Ser-tRNA^(UTu) for up to 20 min. 1.5 μL aliquotstaken at different time points were digested with Nuclease P1 andspotted onto cellulose thin layer chromatography plates Afterdevelopment the plates were analyzed by autoradiography. WhileSer-tRNA^(Sec) is nearly completely converted only approximately 50% ofSec-tRNA^(UTu) is formed by SelA over a course of 20 min.

TABLE 1 Kinetic Parameters of tRNA^(Sec) and tRNA^(UTu) tRNA K_(M) [μM]K_(cat) [s⁻¹] K_(cat)/K_(M) E. coli tRNA^(Sec) 4.1 ± 1.0 0.02 0.005 E.coli tRNA^(Ser) 5.7 ± 1.4 0.01 0.002 tRNA^(UTu) _(am) 5.1 ± 1.1 0.0120.0023

FIG. 13 shows in vitro conversion of tRNA^(Sec) and tRNA^(UTu) _(amber)by PSTK. 10 M of [α32-P] radiolabled Ser-tRNA^(Sec) and Ser-tRNA^(UTu)were incubated at pH 7.2 in the presence of 5 μM Methanococcusmaripaludis PSTK and 5 mM ATP under anaerobic conditions at 37° C. forup to 25 min. 1.5 μL aliquots taken at different time points weredigested with Nuclease P1 and spotted onto cellulose thin layerchromatography plates. After development the plates were analyzed byautoradiography. Approximately 40% of both, Ser-tRNA^(Sec) andSer-tRNA^(UTu) are converted to phosphoseryl-(Sep-)tRNA by PSTK over acourse of 25 min.

Example 7 Sec Complements MGMT Active Site Residue Cys145

O-6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that canprotect the alkyltransferase-deficient E. coli Δada, Δogt-1 strain fromthe DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine(Christians, et al., PNAS, 93:6124-6128 (1996)). MGMT is capable oftransferring the methyl group from DNA with 06-methylguanine to thewild-type Cys145 and designed Sec145, but not to Ser145 at the activesite in a single turn over reaction. E. coli Dada Dotg-1 cellsexpressing either (MGMT) Cys145, amber145 (Sec/Ser) or Ser145 mutantproteins transformed with tRNA^(UTu) _(amber) were pulsed 3× withN-Methyl-N-nitroso-N′-nitroguanidine. Dilutions of the cell cultureswere plated to indicate cellular protection through active MGMT as shownby growth.

FIG. 13 shows amber145 rescues MGMT enzyme activity while Ser145 isinactive, relative to a Cys145 control.

Example 8 Genetic Optimization Increases tRNA^(UTu) Mediated SecIncorporation

tRNA^(UTu) mediated Sec incorporation into Grx1_(C11amC14S) wasgradually increased by genetic optimization of the expression system.Initially Grx1 was expressed under the control of an IPTG inducible T7promoter. The change from genomically encoded SelA to induciblerecombinant SelA increased the Sec incorporation ratio from 23% to 37%.

A further increase to 49% was obtained by adding T7 controlledMethanococcus janaschii PSTK that was codon optimized for the E. coliexpression host. By adding a second selA copy Sec insertion increased to59% while up to 70% incorporation were obtained after the Grx1 reporterwas expressed independently from SelA and PSTK under the control of anarabinose inducible PBAD promoter (FIG. 14).

1. (canceled)
 2. An isolated nucleic acid comprising a nucleic acidsequence encoding a non-naturally occurring tRNA^(Sec), wherein thenon-naturally occurring tRNA^(Sec) is recognized by SerRS and by EF-Tu,or variants thereof, and when aminoacylated with serine the Ser-tRNA isa substrate for SelA or a variant thereof, wherein the tRNA^(Sec) is avariant of a naturally occurring E. coli tRNA^(Sec), wherein thenon-naturally occurring tRNA^(Sec) comprises a mutated antideterminantelement that allows recognition of Sec-tRNA^(Sec) by EF-Tu, and whereinthe anticodon of the non-naturally occurring tRNA^(Sec) hybridizes to astop codon.
 3. (canceled)
 4. The isolated nucleic acid of claim 2further comprising a heterologous expression control sequence.
 5. Anexpression vector comprising the isolated nucleic acid of claim
 4. 6. Ahost cell comprising the isolated nucleic acid of claim
 2. 7. The hostcell of claim 6 wherein the host cell is a prokaryote, archaeon, oreukaryote.
 8. The host cell of claim 7 wherein the prokaryotic cell isE. coli.
 9. The host cell of claim 8 wherein the endogenous E. coligenes encoding selA, selB, and selC, or combinations thereof have beendeleted or mutated to reduce or prevent expression of SelA, SelB, orSelC protein.
 10. The host cell of claim 6 wherein the host cell expressone or more of the proteins selected from the group consisting of SerRS,EF-Tu, and SelA.
 11. The host cell of claim 6 wherein the host cellexpresses SerRS, EF-Tu, and SelA. 12.-17. (canceled)
 18. A method ofmaking a recombinant selenocysteine containing protein comprisingco-expressing a non-naturally occurring tRNA^(Sec) encoded by thenucleic acid of claim 2 in a host cell also expressing SerRS, EF-Tu, andSelA, with a polynucleotide comprising a codon that hybridizes with theanticodon of the non-naturally occurring tRNA^(Sec).
 19. The method ofclaim 18 wherein the codon of the polynucleotide that hybridizes withthe anticodon of the non-naturally occurring tRNA^(Sec) was substitutedfor a codon encoding a cysteine in a reference sequence at least 90%identical to the polynucleotide sequence.
 20. The method of claim 19wherein the polynucleotide encodes an enzyme, cofactor or an antibody.21. (canceled)
 22. The method of claim 18 further comprising purifyingthe recombinant selenocysteine containing protein by chromatographycomprising an activated thiol sepherose. 23.-26. (canceled)
 27. Theisolated nucleic acid of claim 2, wherein the naturally occurring E.coli tRNA^(Sec) comprises the nucleic acid sequence of SEQ ID NO: 1 or avariant thereof with a different anticodon, and wherein thenon-naturally occurring tRNA^(Sec) comprises at least 85% sequenceidentity to SEQ ID NO:1.
 28. The isolated nucleic acid of claim 27,wherein the antideterminant element comprises the last base pair in theamino acid acceptor stem and the first two base pairs in the T-stem ofthe tRNA^(Sec).
 29. The isolated nucleic acid of claim 30, wherein thenon-naturally occurring tRNA^(Sec) is a variant of an E. coli tRNA^(Sec)wherein one or more base pairs selected from the group consisting ofC7•G66, G49•U65, C50•G64 are mutated.
 30. The isolated nucleic acid ofclaim 29, wherein the non-naturally occurring tRNA^(Sec) is a variant ofthe E. coli tRNA^(Sec) according to SEQ ID NO: 1, wherein one or morenucleotides selected from group consisting of 8, 67, 68, 82, 83, and 84are substituted relative to SEQ ID NO: 1, and wherein the anticodon ofnucleotides 35-37 of SEQ ID NO:1 are CUA (amber), UCA (opal), or UUA(ochre).
 31. A non-naturally occurring tRNA^(Sec) encoded by the nucleicacid of claim
 2. 32. The host cell of claim 6 wherein the nucleic acidis integrated into the host cell's genome.
 33. A tRNA^(Sec) comprisingat least 90% sequence identity to SEQ ID NO:1, wherein one or morenucleotides selected from group consisting of 8, 67, 68, 82, 83, and 84are substituted relative to SEQ ID NO:1, and wherein the anticodon ofnucleotides 35-37 of SEQ ID NO:1 are CUA (amber), UCA (opal), or UUA(ochre) and wherein the tRNA^(Sec) is a substrate for EF-Tu.